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How to Write a Research Paper Introduction (with Examples)

The research paper introduction section, along with the Title and Abstract, can be considered the face of any research paper. The following article is intended to guide you in organizing and writing the research paper introduction for a quality academic article or dissertation.

The research paper introduction aims to present the topic to the reader. A study will only be accepted for publishing if you can ascertain that the available literature cannot answer your research question. So it is important to ensure that you have read important studies on that particular topic, especially those within the last five to ten years, and that they are properly referenced in this section. 1 What should be included in the research paper introduction is decided by what you want to tell readers about the reason behind the research and how you plan to fill the knowledge gap. The best research paper introduction provides a systemic review of existing work and demonstrates additional work that needs to be done. It needs to be brief, captivating, and well-referenced; a well-drafted research paper introduction will help the researcher win half the battle.

The introduction for a research paper is where you set up your topic and approach for the reader. It has several key goals:

• Present your research topic
• Capture reader interest
• Summarize existing research
• Position your own approach
• Define your specific research problem and problem statement
• Highlight the novelty and contributions of the study
• Give an overview of the paper’s structure

The research paper introduction can vary in size and structure depending on whether your paper presents the results of original empirical research or is a review paper. Some research paper introduction examples are only half a page while others are a few pages long. In many cases, the introduction will be shorter than all of the other sections of your paper; its length depends on the size of your paper as a whole.

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Table of Contents

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The introduction in a research paper is placed at the beginning to guide the reader from a broad subject area to the specific topic that your research addresses. They present the following information to the reader

• Scope: The topic covered in the research paper
• Context: Background of your topic
• Importance: Why your research matters in that particular area of research and the industry problem that can be targeted

The research paper introduction conveys a lot of information and can be considered an essential roadmap for the rest of your paper. A good introduction for a research paper is important for the following reasons:

• It stimulates your reader’s interest: A good introduction section can make your readers want to read your paper by capturing their interest. It informs the reader what they are going to learn and helps determine if the topic is of interest to them.
• It helps the reader understand the research background: Without a clear introduction, your readers may feel confused and even struggle when reading your paper. A good research paper introduction will prepare them for the in-depth research to come. It provides you the opportunity to engage with the readers and demonstrate your knowledge and authority on the specific topic.
• It explains why your research paper is worth reading: Your introduction can convey a lot of information to your readers. It introduces the topic, why the topic is important, and how you plan to proceed with your research.
• It helps guide the reader through the rest of the paper: The research paper introduction gives the reader a sense of the nature of the information that will support your arguments and the general organization of the paragraphs that will follow. It offers an overview of what to expect when reading the main body of your paper.

What are the parts of introduction in the research?

A good research paper introduction section should comprise three main elements: 2

• What is known: This sets the stage for your research. It informs the readers of what is known on the subject.
• What is lacking: This is aimed at justifying the reason for carrying out your research. This could involve investigating a new concept or method or building upon previous research.
• What you aim to do: This part briefly states the objectives of your research and its major contributions. Your detailed hypothesis will also form a part of this section.

How to write a research paper introduction?

The first step in writing the research paper introduction is to inform the reader what your topic is and why it’s interesting or important. This is generally accomplished with a strong opening statement. The second step involves establishing the kinds of research that have been done and ending with limitations or gaps in the research that you intend to address. Finally, the research paper introduction clarifies how your own research fits in and what problem it addresses. If your research involved testing hypotheses, these should be stated along with your research question. The hypothesis should be presented in the past tense since it will have been tested by the time you are writing the research paper introduction.

The following key points, with examples, can guide you when writing the research paper introduction section:

• Highlight the importance of the research field or topic
• Describe the background of the topic
• Present an overview of current research on the topic

Example: The inclusion of experiential and competency-based learning has benefitted electronics engineering education. Industry partnerships provide an excellent alternative for students wanting to engage in solving real-world challenges. Industry-academia participation has grown in recent years due to the need for skilled engineers with practical training and specialized expertise. However, from the educational perspective, many activities are needed to incorporate sustainable development goals into the university curricula and consolidate learning innovation in universities.

• Reveal a gap in existing research or oppose an existing assumption
• Formulate the research question

Example: There have been plausible efforts to integrate educational activities in higher education electronics engineering programs. However, very few studies have considered using educational research methods for performance evaluation of competency-based higher engineering education, with a focus on technical and or transversal skills. To remedy the current need for evaluating competencies in STEM fields and providing sustainable development goals in engineering education, in this study, a comparison was drawn between study groups without and with industry partners.

• State the purpose of your study
• Highlight the key characteristics of your study
• Describe important results
• Highlight the novelty of the study.
• Offer a brief overview of the structure of the paper.

Example: The study evaluates the main competency needed in the applied electronics course, which is a fundamental core subject for many electronics engineering undergraduate programs. We compared two groups, without and with an industrial partner, that offered real-world projects to solve during the semester. This comparison can help determine significant differences in both groups in terms of developing subject competency and achieving sustainable development goals.

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You can use the same process to develop each section of your article, and finally your research paper in half the time and without any of the stress.

The purpose of the research paper introduction is to introduce the reader to the problem definition, justify the need for the study, and describe the main theme of the study. The aim is to gain the reader’s attention by providing them with necessary background information and establishing the main purpose and direction of the research.

The length of the research paper introduction can vary across journals and disciplines. While there are no strict word limits for writing the research paper introduction, an ideal length would be one page, with a maximum of 400 words over 1-4 paragraphs. Generally, it is one of the shorter sections of the paper as the reader is assumed to have at least a reasonable knowledge about the topic. 2 For example, for a study evaluating the role of building design in ensuring fire safety, there is no need to discuss definitions and nature of fire in the introduction; you could start by commenting upon the existing practices for fire safety and how your study will add to the existing knowledge and practice.

When deciding what to include in the research paper introduction, the rest of the paper should also be considered. The aim is to introduce the reader smoothly to the topic and facilitate an easy read without much dependency on external sources. 3 Below is a list of elements you can include to prepare a research paper introduction outline and follow it when you are writing the research paper introduction. Topic introduction: This can include key definitions and a brief history of the topic. Research context and background: Offer the readers some general information and then narrow it down to specific aspects. Details of the research you conducted: A brief literature review can be included to support your arguments or line of thought. Rationale for the study: This establishes the relevance of your study and establishes its importance. Importance of your research: The main contributions are highlighted to help establish the novelty of your study Research hypothesis: Introduce your research question and propose an expected outcome. Organization of the paper: Include a short paragraph of 3-4 sentences that highlights your plan for the entire paper

Cite only works that are most relevant to your topic; as a general rule, you can include one to three. Note that readers want to see evidence of original thinking. So it is better to avoid using too many references as it does not leave much room for your personal standpoint to shine through. Citations in your research paper introduction support the key points, and the number of citations depend on the subject matter and the point discussed. If the research paper introduction is too long or overflowing with citations, it is better to cite a few review articles rather than the individual articles summarized in the review. A good point to remember when citing research papers in the introduction section is to include at least one-third of the references in the introduction.

The literature review plays a significant role in the research paper introduction section. A good literature review accomplishes the following: Introduces the topic – Establishes the study’s significance – Provides an overview of the relevant literature – Provides context for the study using literature – Identifies knowledge gaps However, remember to avoid making the following mistakes when writing a research paper introduction: Do not use studies from the literature review to aggressively support your research Avoid direct quoting Do not allow literature review to be the focus of this section. Instead, the literature review should only aid in setting a foundation for the manuscript.

Remember the following key points for writing a good research paper introduction: 4

• Avoid stuffing too much general information: Avoid including what an average reader would know and include only that information related to the problem being addressed in the research paper introduction. For example, when describing a comparative study of non-traditional methods for mechanical design optimization, information related to the traditional methods and differences between traditional and non-traditional methods would not be relevant. In this case, the introduction for the research paper should begin with the state-of-the-art non-traditional methods and methods to evaluate the efficiency of newly developed algorithms.
• Avoid packing too many references: Cite only the required works in your research paper introduction. The other works can be included in the discussion section to strengthen your findings.
• Avoid extensive criticism of previous studies: Avoid being overly critical of earlier studies while setting the rationale for your study. A better place for this would be the Discussion section, where you can highlight the advantages of your method.
• Avoid describing conclusions of the study: When writing a research paper introduction remember not to include the findings of your study. The aim is to let the readers know what question is being answered. The actual answer should only be given in the Results and Discussion section.

To summarize, the research paper introduction section should be brief yet informative. It should convince the reader the need to conduct the study and motivate him to read further. If you’re feeling stuck or unsure, choose trusted AI academic writing assistants like Paperpal to effortlessly craft your research paper introduction and other sections of your research article.

1. Jawaid, S. A., & Jawaid, M. (2019). How to write introduction and discussion. Saudi Journal of Anaesthesia, 13(Suppl 1), S18.

2. Dewan, P., & Gupta, P. (2016). Writing the title, abstract and introduction: Looks matter!. Indian pediatrics, 53, 235-241.

3. Cetin, S., & Hackam, D. J. (2005). An approach to the writing of a scientific Manuscript1. Journal of Surgical Research, 128(2), 165-167.

4. Bavdekar, S. B. (2015). Writing introduction: Laying the foundations of a research paper. Journal of the Association of Physicians of India, 63(7), 44-6.

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Home » Research Paper Introduction – Writing Guide and Examples

Research Paper Introduction – Writing Guide and Examples

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Research Paper Introduction

Research paper introduction is the first section of a research paper that provides an overview of the study, its purpose, and the research question (s) or hypothesis (es) being investigated. It typically includes background information about the topic, a review of previous research in the field, and a statement of the research objectives. The introduction is intended to provide the reader with a clear understanding of the research problem, why it is important, and how the study will contribute to existing knowledge in the field. It also sets the tone for the rest of the paper and helps to establish the author’s credibility and expertise on the subject.

How to Write Research Paper Introduction

Writing an introduction for a research paper can be challenging because it sets the tone for the entire paper. Here are some steps to follow to help you write an effective research paper introduction:

• Start with a hook : Begin your introduction with an attention-grabbing statement, a question, or a surprising fact that will make the reader interested in reading further.
• Provide background information: After the hook, provide background information on the topic. This information should give the reader a general idea of what the topic is about and why it is important.
• State the research problem: Clearly state the research problem or question that the paper addresses. This should be done in a concise and straightforward manner.
• State the research objectives: After stating the research problem, clearly state the research objectives. This will give the reader an idea of what the paper aims to achieve.
• Provide a brief overview of the paper: At the end of the introduction, provide a brief overview of the paper. This should include a summary of the main points that will be discussed in the paper.
• Revise and refine: Finally, revise and refine your introduction to ensure that it is clear, concise, and engaging.

Structure of Research Paper Introduction

The following is a typical structure for a research paper introduction:

• Background Information: This section provides an overview of the topic of the research paper, including relevant background information and any previous research that has been done on the topic. It helps to give the reader a sense of the context for the study.
• Problem Statement: This section identifies the specific problem or issue that the research paper is addressing. It should be clear and concise, and it should articulate the gap in knowledge that the study aims to fill.
• Research Question/Hypothesis : This section states the research question or hypothesis that the study aims to answer. It should be specific and focused, and it should clearly connect to the problem statement.
• Significance of the Study: This section explains why the research is important and what the potential implications of the study are. It should highlight the contribution that the research makes to the field.
• Methodology: This section describes the research methods that were used to conduct the study. It should be detailed enough to allow the reader to understand how the study was conducted and to evaluate the validity of the results.
• Organization of the Paper : This section provides a brief overview of the structure of the research paper. It should give the reader a sense of what to expect in each section of the paper.

Research Paper Introduction Examples

Research Paper Introduction Examples could be:

Example 1: In recent years, the use of artificial intelligence (AI) has become increasingly prevalent in various industries, including healthcare. AI algorithms are being developed to assist with medical diagnoses, treatment recommendations, and patient monitoring. However, as the use of AI in healthcare grows, ethical concerns regarding privacy, bias, and accountability have emerged. This paper aims to explore the ethical implications of AI in healthcare and propose recommendations for addressing these concerns.

Example 2: Climate change is one of the most pressing issues facing our planet today. The increasing concentration of greenhouse gases in the atmosphere has resulted in rising temperatures, changing weather patterns, and other environmental impacts. In this paper, we will review the scientific evidence on climate change, discuss the potential consequences of inaction, and propose solutions for mitigating its effects.

Example 3: The rise of social media has transformed the way we communicate and interact with each other. While social media platforms offer many benefits, including increased connectivity and access to information, they also present numerous challenges. In this paper, we will examine the impact of social media on mental health, privacy, and democracy, and propose solutions for addressing these issues.

Example 4: The use of renewable energy sources has become increasingly important in the face of climate change and environmental degradation. While renewable energy technologies offer many benefits, including reduced greenhouse gas emissions and energy independence, they also present numerous challenges. In this paper, we will assess the current state of renewable energy technology, discuss the economic and political barriers to its adoption, and propose solutions for promoting the widespread use of renewable energy.

Purpose of Research Paper Introduction

The introduction section of a research paper serves several important purposes, including:

• Providing context: The introduction should give readers a general understanding of the topic, including its background, significance, and relevance to the field.
• Presenting the research question or problem: The introduction should clearly state the research question or problem that the paper aims to address. This helps readers understand the purpose of the study and what the author hopes to accomplish.
• Reviewing the literature: The introduction should summarize the current state of knowledge on the topic, highlighting the gaps and limitations in existing research. This shows readers why the study is important and necessary.
• Outlining the scope and objectives of the study: The introduction should describe the scope and objectives of the study, including what aspects of the topic will be covered, what data will be collected, and what methods will be used.
• Previewing the main findings and conclusions : The introduction should provide a brief overview of the main findings and conclusions that the study will present. This helps readers anticipate what they can expect to learn from the paper.

When to Write Research Paper Introduction

The introduction of a research paper is typically written after the research has been conducted and the data has been analyzed. This is because the introduction should provide an overview of the research problem, the purpose of the study, and the research questions or hypotheses that will be investigated.

Once you have a clear understanding of the research problem and the questions that you want to explore, you can begin to write the introduction. It’s important to keep in mind that the introduction should be written in a way that engages the reader and provides a clear rationale for the study. It should also provide context for the research by reviewing relevant literature and explaining how the study fits into the larger field of research.

Advantages of Research Paper Introduction

The introduction of a research paper has several advantages, including:

• Establishing the purpose of the research: The introduction provides an overview of the research problem, question, or hypothesis, and the objectives of the study. This helps to clarify the purpose of the research and provide a roadmap for the reader to follow.
• Providing background information: The introduction also provides background information on the topic, including a review of relevant literature and research. This helps the reader understand the context of the study and how it fits into the broader field of research.
• Demonstrating the significance of the research: The introduction also explains why the research is important and relevant. This helps the reader understand the value of the study and why it is worth reading.
• Setting expectations: The introduction sets the tone for the rest of the paper and prepares the reader for what is to come. This helps the reader understand what to expect and how to approach the paper.
• Grabbing the reader’s attention: A well-written introduction can grab the reader’s attention and make them interested in reading further. This is important because it can help to keep the reader engaged and motivated to read the rest of the paper.
• Creating a strong first impression: The introduction is the first part of the research paper that the reader will see, and it can create a strong first impression. A well-written introduction can make the reader more likely to take the research seriously and view it as credible.
• Establishing the author’s credibility: The introduction can also establish the author’s credibility as a researcher. By providing a clear and thorough overview of the research problem and relevant literature, the author can demonstrate their expertise and knowledge in the field.
• Providing a structure for the paper: The introduction can also provide a structure for the rest of the paper. By outlining the main sections and sub-sections of the paper, the introduction can help the reader navigate the paper and find the information they are looking for.

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Research Paper Introduction Examples

Looking for research paper introduction examples? Quotes, anecdotes, questions, examples, and broad statements—all of them can be used successfully to write an introduction for a research paper. It’s instructive to see them in action, in the hands of skilled academic writers.

Let’s begin with David M. Kennedy’s superb history, Freedom from Fear: The American People in Depression and War, 1929–1945 . Kennedy begins each chapter with a quote, followed by his text. The quote above chapter 1 shows President Hoover speaking in 1928 about America’s golden future. The text below it begins with the stock market collapse of 1929. It is a riveting account of just how wrong Hoover was. The text about the Depression is stronger because it contrasts so starkly with the optimistic quotation.

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“We in America today are nearer the final triumph over poverty than ever before in the history of any land.”—Herbert Hoover, August 11, 1928 Like an earthquake, the stock market crash of October 1929 cracked startlingly across the United States, the herald of a crisis that was to shake the American way of life to its foundations. The events of the ensuing decade opened a fissure across the landscape of American history no less gaping than that opened by the volley on Lexington Common in April 1775 or by the bombardment of Sumter on another April four score and six years later. (adsbygoogle = window.adsbygoogle || []).push({}); The ratcheting ticker machines in the autumn of 1929 did not merely record avalanching stock prices. In time they came also to symbolize the end of an era. (David M. Kennedy, Freedom from Fear: The American People in Depression and War, 1929–1945 . New York: Oxford University Press, 1999, p. 10)

Kennedy has exciting, wrenching material to work with. John Mueller faces the exact opposite problem. In Retreat from Doomsday: The Obsolescence of Major War , he is trying to explain why Great Powers have suddenly stopped fighting each other. For centuries they made war on each other with devastating regularity, killing millions in the process. But now, Mueller thinks, they have not just paused; they have stopped permanently. He is literally trying to explain why “nothing is happening now.” That may be an exciting topic intellectually, it may have great practical significance, but “nothing happened” is not a very promising subject for an exciting opening paragraph. Mueller manages to make it exciting and, at the same time, shows why it matters so much. Here’s his opening, aptly entitled “History’s Greatest Nonevent”:

On May 15, 1984, the major countries of the developed world had managed to remain at peace with each other for the longest continuous stretch of time since the days of the Roman Empire. If a significant battle in a war had been fought on that day, the press would have bristled with it. As usual, however, a landmark crossing in the history of peace caused no stir: the most prominent story in the New York Times that day concerned the saga of a manicurist, a machinist, and a cleaning woman who had just won a big Lotto contest. This book seeks to develop an explanation for what is probably the greatest nonevent in human history. (John Mueller, Retreat from Doomsday: The Obsolescence of Major War . New York: Basic Books, 1989, p. 3)

In the space of a few sentences, Mueller sets up his puzzle and reveals its profound human significance. At the same time, he shows just how easy it is to miss this milestone in the buzz of daily events. Notice how concretely he does that. He doesn’t just say that the New York Times ignored this record setting peace. He offers telling details about what they covered instead: “a manicurist, a machinist, and a cleaning woman who had just won a big Lotto contest.” Likewise, David Kennedy immediately entangles us in concrete events: the stunning stock market crash of 1929. These are powerful openings that capture readers’ interests, establish puzzles, and launch narratives.

Sociologist James Coleman begins in a completely different way, by posing the basic questions he will study. His ambitious book, Foundations of Social Theory , develops a comprehensive theory of social life, so it is entirely appropriate for him to begin with some major questions. But he could just as easily have begun with a compelling story or anecdote. He includes many of them elsewhere in his book. His choice for the opening, though, is to state his major themes plainly and frame them as a paradox. Sociologists, he says, are interested in aggregate behavior—how people act in groups, organizations, or large numbers—yet they mostly examine individuals:

A central problem in social science is that of accounting for the function of some kind of social system. Yet in most social research, observations are not made on the system as a whole, but on some part of it. In fact, the natural unit of observation is the individual person…  This has led to a widening gap between theory and research… (James S. Coleman, Foundations of Social Theory . Cambridge, MA: Harvard University Press, 1990, pp. 1–2)

After expanding on this point, Coleman explains that he will not try to remedy the problem by looking solely at groups or aggregate-level data. That’s a false solution, he says, because aggregates don’t act; individuals do. So the real problem is to show the links between individual actions and aggregate outcomes, between the micro and the macro.

The major problem for explanations of system behavior based on actions and orientations at a level below that of the system [in this case, on individual-level actions] is that of moving from the lower level to the system level. This has been called the micro-to-macro problem, and it is pervasive throughout the social sciences. (Coleman, Foundations of Social Theory , p. 6)

Explaining how to deal with this “micro-to-macro problem” is the central issue of Coleman’s book, and he announces it at the beginning.

Coleman’s theory-driven opening stands at the opposite end of the spectrum from engaging stories or anecdotes, which are designed to lure the reader into the narrative and ease the path to a more analytic treatment later in the text. Take, for example, the opening sentences of Robert L. Herbert’s sweeping study Impressionism: Art, Leisure, and Parisian Society : “When Henry Tuckerman came to Paris in 1867, one of the thousands of Americans attracted there by the huge international exposition, he was bowled over by the extraordinary changes since his previous visit twenty years before.” (Robert L. Herbert, Impressionism: Art, Leisure, and Parisian Society . New Haven, CT: Yale University Press, 1988, p. 1.) Herbert fills in the evocative details to set the stage for his analysis of the emerging Impressionist art movement and its connection to Parisian society and leisure in this period.

David Bromwich writes about Wordsworth, a poet so familiar to students of English literature that it is hard to see him afresh, before his great achievements, when he was just a young outsider starting to write. To draw us into Wordsworth’s early work, Bromwich wants us to set aside our entrenched images of the famous mature poet and see him as he was in the 1790s, as a beginning writer on the margins of society. He accomplishes this ambitious task in the opening sentences of Disowned by Memory: Wordsworth’s Poetry of the 1790s :

Wordsworth turned to poetry after the revolution to remind himself that he was still a human being. It was a curious solution, to a difficulty many would not have felt. The whole interest of his predicament is that he did feel it. Yet Wordsworth is now so established an eminence—his name so firmly fixed with readers as a moralist of self-trust emanating from complete self-security—that it may seem perverse to imagine him as a criminal seeking expiation. Still, that is a picture we get from The Borderers and, at a longer distance, from “Tintern Abbey.” (David Bromwich, Disowned by Memory: Wordsworth’s Poetry of the 1790s . Chicago: University of Chicago Press, 1998, p. 1)

That’s a wonderful opening! Look at how much Bromwich accomplishes in just a few words. He not only prepares the way for analyzing Wordsworth’s early poetry; he juxtaposes the anguished young man who wrote it to the self-confident, distinguished figure he became—the eminent man we can’t help remembering as we read his early poetry.

Let us highlight a couple of other points in this passage because they illustrate some intelligent writing choices. First, look at the odd comma in this sentence: “It was a curious solution, to a difficulty many would not have felt.” Any standard grammar book would say that comma is wrong and should be omitted. Why did Bromwich insert it? Because he’s a fine writer, thinking of his sentence rhythm and the point he wants to make. The comma does exactly what it should. It makes us pause, breaking the sentence into two parts, each with an interesting point. One is that Wordsworth felt a difficulty others would not have; the other is that he solved it in a distinctive way. It would be easy for readers to glide over this double message, so Bromwich has inserted a speed bump to slow us down. Most of the time, you should follow grammatical rules, like those about commas, but you should bend them when it serves a good purpose. That’s what the writer does here.

The second small point is the phrase “after the revolution” in the first sentence: “Wordsworth turned to poetry after the revolution to remind himself that he was still a human being.” Why doesn’t Bromwich say “after the French Revolution”? Because he has judged his book’s audience. He is writing for specialists who already know which revolution is reverberating through English life in the 1790s. It is the French Revolution, not the earlier loss of the American colonies. If Bromwich were writing for a much broader audience—say, the New York Times Book Review—he would probably insert the extra word to avoid confusion.

The message “Know your audience” applies to all writers. Don’t talk down to them by assuming they can’t get dressed in the morning. Don’t strut around showing off your book learnin’ by tossing in arcane facts and esoteric language for its own sake. Neither will win over readers.

Bromwich, Herbert, and Coleman open their works in different ways, but their choices work well for their different texts. Your task is to decide what kind of opening will work best for yours. Don’t let that happen by default, by grabbing the first idea you happen upon. Consider a couple of different ways of opening your thesis and then choose the one you prefer. Give yourself some options, think them over, then make an informed choice.

Using the Introduction to Map out Your Writing

Whether you begin with a story, puzzle, or broad statement, the next part of the research paper introduction should pose your main questions and establish your argument. This is your thesis statement—your viewpoint along with the supporting reasons and evidence. It should be articulated plainly so readers understand full well what your paper is about and what it will argue.

After that, give your readers a road map of what’s to come. That’s normally done at the end of the introductory section (or, in a book, at the end of the introductory chapter). Here’s John J. Mearsheimer presenting such a road map in The Tragedy of Great Power Politics . He not only tells us the order of upcoming chapters, he explains why he’s chosen that order and which chapters are most important:

The Plan of the Book The rest of the chapters in this book are concerned mainly with answering the six big questions about power which I identified earlier. Chapter 2, which is probably the most important chapter in the book, lays out my theory of why states compete for power and why they pursue hegemony. In Chapters 3 and 4, I define power and explain how to measure it. I do this in order to lay the groundwork for testing my theory… (John J. Mearsheimer, The Tragedy of Great Power Politics . New York: W. W. Norton, 2001, p. 27)

As this excerpt makes clear, Mearsheimer has already laid out his “six big questions” in the research paper introduction. Now he’s showing us the path ahead, the path to answering those questions.

At the end of the research paper introduction, give your readers a road map of what’s to come. Tell them what the upcoming sections will be and why they are arranged in this particular order.

Learn how to write an introduction for a research paper .

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Organizing Your Social Sciences Research Paper

• 4. The Introduction
• Purpose of Guide
• Design Flaws to Avoid
• Independent and Dependent Variables
• Glossary of Research Terms
• Reading Research Effectively
• Narrowing a Topic Idea
• Broadening a Topic Idea
• Extending the Timeliness of a Topic Idea
• Academic Writing Style
• Applying Critical Thinking
• Choosing a Title
• Making an Outline
• Paragraph Development
• Research Process Video Series
• Executive Summary
• The C.A.R.S. Model
• Background Information
• The Research Problem/Question
• Theoretical Framework
• Citation Tracking
• Content Alert Services
• Evaluating Sources
• Primary Sources
• Secondary Sources
• Tiertiary Sources
• Scholarly vs. Popular Publications
• Qualitative Methods
• Quantitative Methods
• Insiderness
• Using Non-Textual Elements
• Limitations of the Study
• Common Grammar Mistakes
• Writing Concisely
• Avoiding Plagiarism
• Footnotes or Endnotes?
• Further Readings
• Generative AI and Writing
• USC Libraries Tutorials and Other Guides
• Bibliography

The introduction leads the reader from a general subject area to a particular topic of inquiry. It establishes the scope, context, and significance of the research being conducted by summarizing current understanding and background information about the topic, stating the purpose of the work in the form of the research problem supported by a hypothesis or a set of questions, explaining briefly the methodological approach used to examine the research problem, highlighting the potential outcomes your study can reveal, and outlining the remaining structure and organization of the paper.

Key Elements of the Research Proposal. Prepared under the direction of the Superintendent and by the 2010 Curriculum Design and Writing Team. Baltimore County Public Schools.

Importance of a Good Introduction

Think of the introduction as a mental road map that must answer for the reader these four questions:

• What was I studying?
• Why was this topic important to investigate?
• What did we know about this topic before I did this study?
• How will this study advance new knowledge or new ways of understanding?

According to Reyes, there are three overarching goals of a good introduction: 1) ensure that you summarize prior studies about the topic in a manner that lays a foundation for understanding the research problem; 2) explain how your study specifically addresses gaps in the literature, insufficient consideration of the topic, or other deficiency in the literature; and, 3) note the broader theoretical, empirical, and/or policy contributions and implications of your research.

A well-written introduction is important because, quite simply, you never get a second chance to make a good first impression. The opening paragraphs of your paper will provide your readers with their initial impressions about the logic of your argument, your writing style, the overall quality of your research, and, ultimately, the validity of your findings and conclusions. A vague, disorganized, or error-filled introduction will create a negative impression, whereas, a concise, engaging, and well-written introduction will lead your readers to think highly of your analytical skills, your writing style, and your research approach. All introductions should conclude with a brief paragraph that describes the organization of the rest of the paper.

Hirano, Eliana. “Research Article Introductions in English for Specific Purposes: A Comparison between Brazilian, Portuguese, and English.” English for Specific Purposes 28 (October 2009): 240-250; Samraj, B. “Introductions in Research Articles: Variations Across Disciplines.” English for Specific Purposes 21 (2002): 1–17; Introductions. The Writing Center. University of North Carolina; “Writing Introductions.” In Good Essay Writing: A Social Sciences Guide. Peter Redman. 4th edition. (London: Sage, 2011), pp. 63-70; Reyes, Victoria. Demystifying the Journal Article. Inside Higher Education.

Structure and Writing Style

I.  Structure and Approach

The introduction is the broad beginning of the paper that answers three important questions for the reader:

• What is this?
• Why should I read it?
• What do you want me to think about / consider doing / react to?

Think of the structure of the introduction as an inverted triangle of information that lays a foundation for understanding the research problem. Organize the information so as to present the more general aspects of the topic early in the introduction, then narrow your analysis to more specific topical information that provides context, finally arriving at your research problem and the rationale for studying it [often written as a series of key questions to be addressed or framed as a hypothesis or set of assumptions to be tested] and, whenever possible, a description of the potential outcomes your study can reveal.

These are general phases associated with writing an introduction: 1.  Establish an area to research by:

• Highlighting the importance of the topic, and/or
• Making general statements about the topic, and/or
• Presenting an overview on current research on the subject.

2.  Identify a research niche by:

• Opposing an existing assumption, and/or
• Revealing a gap in existing research, and/or
• Formulating a research question or problem, and/or
• Continuing a disciplinary tradition.

3.  Place your research within the research niche by:

• Stating the intent of your study,
• Outlining the key characteristics of your study,
• Describing important results, and
• Giving a brief overview of the structure of the paper.

NOTE:   It is often useful to review the introduction late in the writing process. This is appropriate because outcomes are unknown until you've completed the study. After you complete writing the body of the paper, go back and review introductory descriptions of the structure of the paper, the method of data gathering, the reporting and analysis of results, and the conclusion. Reviewing and, if necessary, rewriting the introduction ensures that it correctly matches the overall structure of your final paper.

II.  Delimitations of the Study

Delimitations refer to those characteristics that limit the scope and define the conceptual boundaries of your research . This is determined by the conscious exclusionary and inclusionary decisions you make about how to investigate the research problem. In other words, not only should you tell the reader what it is you are studying and why, but you must also acknowledge why you rejected alternative approaches that could have been used to examine the topic.

Obviously, the first limiting step was the choice of research problem itself. However, implicit are other, related problems that could have been chosen but were rejected. These should be noted in the conclusion of your introduction. For example, a delimitating statement could read, "Although many factors can be understood to impact the likelihood young people will vote, this study will focus on socioeconomic factors related to the need to work full-time while in school." The point is not to document every possible delimiting factor, but to highlight why previously researched issues related to the topic were not addressed.

Examples of delimitating choices would be:

• The key aims and objectives of your study,
• The research questions that you address,
• The variables of interest [i.e., the various factors and features of the phenomenon being studied],
• The method(s) of investigation,
• The time period your study covers, and
• Any relevant alternative theoretical frameworks that could have been adopted.

Review each of these decisions. Not only do you clearly establish what you intend to accomplish in your research, but you should also include a declaration of what the study does not intend to cover. In the latter case, your exclusionary decisions should be based upon criteria understood as, "not interesting"; "not directly relevant"; “too problematic because..."; "not feasible," and the like. Make this reasoning explicit!

NOTE:   Delimitations refer to the initial choices made about the broader, overall design of your study and should not be confused with documenting the limitations of your study discovered after the research has been completed.

ANOTHER NOTE: Do not view delimitating statements as admitting to an inherent failing or shortcoming in your research. They are an accepted element of academic writing intended to keep the reader focused on the research problem by explicitly defining the conceptual boundaries and scope of your study. It addresses any critical questions in the reader's mind of, "Why the hell didn't the author examine this?"

III.  The Narrative Flow

Issues to keep in mind that will help the narrative flow in your introduction :

• Your introduction should clearly identify the subject area of interest . A simple strategy to follow is to use key words from your title in the first few sentences of the introduction. This will help focus the introduction on the topic at the appropriate level and ensures that you get to the subject matter quickly without losing focus, or discussing information that is too general.
• Establish context by providing a brief and balanced review of the pertinent published literature that is available on the subject. The key is to summarize for the reader what is known about the specific research problem before you did your analysis. This part of your introduction should not represent a comprehensive literature review--that comes next. It consists of a general review of the important, foundational research literature [with citations] that establishes a foundation for understanding key elements of the research problem. See the drop-down menu under this tab for " Background Information " regarding types of contexts.
• Clearly state the hypothesis that you investigated . When you are first learning to write in this format it is okay, and actually preferable, to use a past statement like, "The purpose of this study was to...." or "We investigated three possible mechanisms to explain the...."
• Why did you choose this kind of research study or design? Provide a clear statement of the rationale for your approach to the problem studied. This will usually follow your statement of purpose in the last paragraph of the introduction.

IV.  Engaging the Reader

A research problem in the social sciences can come across as dry and uninteresting to anyone unfamiliar with the topic . Therefore, one of the goals of your introduction is to make readers want to read your paper. Here are several strategies you can use to grab the reader's attention:

• Open with a compelling story . Almost all research problems in the social sciences, no matter how obscure or esoteric , are really about the lives of people. Telling a story that humanizes an issue can help illuminate the significance of the problem and help the reader empathize with those affected by the condition being studied.
• Include a strong quotation or a vivid, perhaps unexpected, anecdote . During your review of the literature, make note of any quotes or anecdotes that grab your attention because they can used in your introduction to highlight the research problem in a captivating way.
• Pose a provocative or thought-provoking question . Your research problem should be framed by a set of questions to be addressed or hypotheses to be tested. However, a provocative question can be presented in the beginning of your introduction that challenges an existing assumption or compels the reader to consider an alternative viewpoint that helps establish the significance of your study. 
• Describe a puzzling scenario or incongruity . This involves highlighting an interesting quandary concerning the research problem or describing contradictory findings from prior studies about a topic. Posing what is essentially an unresolved intellectual riddle about the problem can engage the reader's interest in the study.
• Cite a stirring example or case study that illustrates why the research problem is important . Draw upon the findings of others to demonstrate the significance of the problem and to describe how your study builds upon or offers alternatives ways of investigating this prior research.

NOTE:   It is important that you choose only one of the suggested strategies for engaging your readers. This avoids giving an impression that your paper is more flash than substance and does not distract from the substance of your study.

Freedman, Leora  and Jerry Plotnick. Introductions and Conclusions. University College Writing Centre. University of Toronto; Introduction. The Structure, Format, Content, and Style of a Journal-Style Scientific Paper. Department of Biology. Bates College; Introductions. The Writing Center. University of North Carolina; Introductions. The Writer’s Handbook. Writing Center. University of Wisconsin, Madison; Introductions, Body Paragraphs, and Conclusions for an Argument Paper. The Writing Lab and The OWL. Purdue University; “Writing Introductions.” In Good Essay Writing: A Social Sciences Guide . Peter Redman. 4th edition. (London: Sage, 2011), pp. 63-70; Resources for Writers: Introduction Strategies. Program in Writing and Humanistic Studies. Massachusetts Institute of Technology; Sharpling, Gerald. Writing an Introduction. Centre for Applied Linguistics, University of Warwick; Samraj, B. “Introductions in Research Articles: Variations Across Disciplines.” English for Specific Purposes 21 (2002): 1–17; Swales, John and Christine B. Feak. Academic Writing for Graduate Students: Essential Skills and Tasks . 2nd edition. Ann Arbor, MI: University of Michigan Press, 2004 ; Writing Your Introduction. Department of English Writing Guide. George Mason University.

Writing Tip

Avoid the "Dictionary" Introduction

Giving the dictionary definition of words related to the research problem may appear appropriate because it is important to define specific terminology that readers may be unfamiliar with. However, anyone can look a word up in the dictionary and a general dictionary is not a particularly authoritative source because it doesn't take into account the context of your topic and doesn't offer particularly detailed information. Also, placed in the context of a particular discipline, a term or concept may have a different meaning than what is found in a general dictionary. If you feel that you must seek out an authoritative definition, use a subject specific dictionary or encyclopedia [e.g., if you are a sociology student, search for dictionaries of sociology]. A good database for obtaining definitive definitions of concepts or terms is Credo Reference .

Saba, Robert. The College Research Paper. Florida International University; Introductions. The Writing Center. University of North Carolina.

Another Writing Tip

When Do I Begin?

A common question asked at the start of any paper is, "Where should I begin?" An equally important question to ask yourself is, "When do I begin?" Research problems in the social sciences rarely rest in isolation from history. Therefore, it is important to lay a foundation for understanding the historical context underpinning the research problem. However, this information should be brief and succinct and begin at a point in time that illustrates the study's overall importance. For example, a study that investigates coffee cultivation and export in West Africa as a key stimulus for local economic growth needs to describe the beginning of exporting coffee in the region and establishing why economic growth is important. You do not need to give a long historical explanation about coffee exports in Africa. If a research problem requires a substantial exploration of the historical context, do this in the literature review section. In your introduction, make note of this as part of the "roadmap" [see below] that you use to describe the organization of your paper.

Introductions. The Writing Center. University of North Carolina; “Writing Introductions.” In Good Essay Writing: A Social Sciences Guide . Peter Redman. 4th edition. (London: Sage, 2011), pp. 63-70.

Yet Another Writing Tip

Always End with a Roadmap

The final paragraph or sentences of your introduction should forecast your main arguments and conclusions and provide a brief description of the rest of the paper [the "roadmap"] that let's the reader know where you are going and what to expect. A roadmap is important because it helps the reader place the research problem within the context of their own perspectives about the topic. In addition, concluding your introduction with an explicit roadmap tells the reader that you have a clear understanding of the structural purpose of your paper. In this way, the roadmap acts as a type of promise to yourself and to your readers that you will follow a consistent and coherent approach to addressing the topic of inquiry. Refer to it often to help keep your writing focused and organized.

Cassuto, Leonard. “On the Dissertation: How to Write the Introduction.” The Chronicle of Higher Education , May 28, 2018; Radich, Michael. A Student's Guide to Writing in East Asian Studies . (Cambridge, MA: Harvard University Writing n. d.), pp. 35-37.

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How to Write a Research Introduction

Last Updated: December 6, 2023 Fact Checked

This article was co-authored by Megan Morgan, PhD . Megan Morgan is a Graduate Program Academic Advisor in the School of Public & International Affairs at the University of Georgia. She earned her PhD in English from the University of Georgia in 2015. There are 7 references cited in this article, which can be found at the bottom of the page. This article has been fact-checked, ensuring the accuracy of any cited facts and confirming the authority of its sources. This article has been viewed 2,653,863 times.

The introduction to a research paper can be the most challenging part of the paper to write. The length of the introduction will vary depending on the type of research paper you are writing. An introduction should announce your topic, provide context and a rationale for your work, before stating your research questions and hypothesis. Well-written introductions set the tone for the paper, catch the reader's interest, and communicate the hypothesis or thesis statement.

Introducing the Topic of the Paper

• In scientific papers this is sometimes known as an "inverted triangle", where you start with the broadest material at the start, before zooming in on the specifics. [2] X Research source
• The sentence "Throughout the 20th century, our views of life on other planets have drastically changed" introduces a topic, but does so in broad terms.
• It provides the reader with an indication of the content of the essay and encourages them to read on.

• For example, if you were writing a paper about the behaviour of mice when exposed to a particular substance, you would include the word "mice", and the scientific name of the relevant compound in the first sentences.
• If you were writing a history paper about the impact of the First World War on gender relations in Britain, you should mention those key words in your first few lines.

• This is especially important if you are attempting to develop a new conceptualization that uses language and terminology your readers may be unfamiliar with.

• If you use an anecdote ensure that is short and highly relevant for your research. It has to function in the same way as an alternative opening, namely to announce the topic of your research paper to your reader.
• For example, if you were writing a sociology paper about re-offending rates among young offenders, you could include a brief story of one person whose story reflects and introduces your topic.
• This kind of approach is generally not appropriate for the introduction to a natural or physical sciences research paper where the writing conventions are different.

Establishing the Context for Your Paper

• It is important to be concise in the introduction, so provide an overview on recent developments in the primary research rather than a lengthy discussion.
• You can follow the "inverted triangle" principle to focus in from the broader themes to those to which you are making a direct contribution with your paper.
• A strong literature review presents important background information to your own research and indicates the importance of the field.

• By making clear reference to existing work you can demonstrate explicitly the specific contribution you are making to move the field forward.
• You can identify a gap in the existing scholarship and explain how you are addressing it and moving understanding forward.

• For example, if you are writing a scientific paper you could stress the merits of the experimental approach or models you have used.
• Stress what is novel in your research and the significance of your new approach, but don't give too much detail in the introduction.
• A stated rationale could be something like: "the study evaluates the previously unknown anti-inflammatory effects of a topical compound in order to evaluate its potential clinical uses".

Specifying Your Research Questions and Hypothesis

• The research question or questions generally come towards the end of the introduction, and should be concise and closely focused.
• The research question might recall some of the key words established in the first few sentences and the title of your paper.
• An example of a research question could be "what were the consequences of the North American Free Trade Agreement on the Mexican export economy?"
• This could be honed further to be specific by referring to a particular element of the Free Trade Agreement and the impact on a particular industry in Mexico, such as clothing manufacture.
• A good research question should shape a problem into a testable hypothesis.

• If possible try to avoid using the word "hypothesis" and rather make this implicit in your writing. This can make your writing appear less formulaic.
• In a scientific paper, giving a clear one-sentence overview of your results and their relation to your hypothesis makes the information clear and accessible. [10] X Trustworthy Source PubMed Central Journal archive from the U.S. National Institutes of Health Go to source
• An example of a hypothesis could be "mice deprived of food for the duration of the study were expected to become more lethargic than those fed normally".

• This is not always necessary and you should pay attention to the writing conventions in your discipline.
• In a natural sciences paper, for example, there is a fairly rigid structure which you will be following.
• A humanities or social science paper will most likely present more opportunities to deviate in how you structure your paper.

Research Introduction Help

Community Q&A

• Use your research papers' outline to help you decide what information to include when writing an introduction. Thanks Helpful 0 Not Helpful 1
• Consider drafting your introduction after you have already completed the rest of your research paper. Writing introductions last can help ensure that you don't leave out any major points. Thanks Helpful 0 Not Helpful 0

• Avoid emotional or sensational introductions; these can create distrust in the reader. Thanks Helpful 50 Not Helpful 12
• Generally avoid using personal pronouns in your introduction, such as "I," "me," "we," "us," "my," "mine," or "our." Thanks Helpful 31 Not Helpful 7
• Don't overwhelm the reader with an over-abundance of information. Keep the introduction as concise as possible by saving specific details for the body of your paper. Thanks Helpful 24 Not Helpful 14

You Might Also Like

• ↑ https://library.sacredheart.edu/c.php?g=29803&p=185916
• ↑ https://www.aresearchguide.com/inverted-pyramid-structure-in-writing.html
• ↑ https://libguides.usc.edu/writingguide/introduction
• ↑ https://writing.wisc.edu/Handbook/PlanResearchPaper.html
• ↑ https://dept.writing.wisc.edu/wac/writing-an-introduction-for-a-scientific-paper/
• ↑ https://writing.wisc.edu/handbook/assignments/planresearchpaper/
• ↑ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178846/

About This Article

To introduce your research paper, use the first 1-2 sentences to describe your general topic, such as “women in World War I.” Include and define keywords, such as “gender relations,” to show your reader where you’re going. Mention previous research into the topic with a phrase like, “Others have studied…”, then transition into what your contribution will be and why it’s necessary. Finally, state the questions that your paper will address and propose your “answer” to them as your thesis statement. For more information from our English Ph.D. co-author about how to craft a strong hypothesis and thesis, keep reading! Did this summary help you? Yes No

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Microsoft 365 Life Hacks > Writing > How to write an introduction for a research paper

How to write an introduction for a research paper

Beginnings are hard. Beginning a research paper is no exception. Many students—and pros—struggle with how to write an introduction for a research paper.

This short guide will describe the purpose of a research paper introduction and how to create a good one.

What is an introduction for a research paper?

Introductions to research papers do a lot of work.

It may seem obvious, but introductions are always placed at the beginning of a paper. They guide your reader from a general subject area to the narrow topic that your paper covers. They also explain your paper’s:

• Scope: The topic you’ll be covering
• Context: The background of your topic
• Importance: Why your research matters in the context of an industry or the world

Your introduction will cover a lot of ground. However, it will only be half of a page to a few pages long. The length depends on the size of your paper as a whole. In many cases, the introduction will be shorter than all of the other sections of your paper.

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Why is an introduction vital to a research paper?

The introduction to your research paper isn’t just important. It’s critical.

Your readers don’t know what your research paper is about from the title. That’s where your introduction comes in. A good introduction will:

• Help your reader understand your topic’s background
• Explain why your research paper is worth reading
• Offer a guide for navigating the rest of the piece
• Pique your reader’s interest

Without a clear introduction, your readers will struggle. They may feel confused when they start reading your paper. They might even give up entirely. Your introduction will ground them and prepare them for the in-depth research to come.

What should you include in an introduction for a research paper?

Research paper introductions are always unique. After all, research is original by definition. However, they often contain six essential items. These are:

• An overview of the topic. Start with a general overview of your topic. Narrow the overview until you address your paper’s specific subject. Then, mention questions or concerns you had about the case. Note that you will address them in the publication.
• Prior research. Your introduction is the place to review other conclusions on your topic. Include both older scholars and modern scholars. This background information shows that you are aware of prior research. It also introduces past findings to those who might not have that expertise.
• A rationale for your paper. Explain why your topic needs to be addressed right now. If applicable, connect it to current issues. Additionally, you can show a problem with former theories or reveal a gap in current research. No matter how you do it, a good rationale will interest your readers and demonstrate why they must read the rest of your paper.
• Describe the methodology you used. Recount your processes to make your paper more credible. Lay out your goal and the questions you will address. Reveal how you conducted research and describe how you measured results. Moreover, explain why you made key choices.
• A thesis statement. Your main introduction should end with a thesis statement. This statement summarizes the ideas that will run through your entire research article. It should be straightforward and clear.
• An outline. Introductions often conclude with an outline. Your layout should quickly review what you intend to cover in the following sections. Think of it as a roadmap, guiding your reader to the end of your paper.

These six items are emphasized more or less, depending on your field. For example, a physics research paper might emphasize methodology. An English journal article might highlight the overview.

Three tips for writing your introduction

We don’t just want you to learn how to write an introduction for a research paper. We want you to learn how to make it shine.

There are three things you can do that will make it easier to write a great introduction. You can:

• Write your introduction last. An introduction summarizes all of the things you’ve learned from your research. While it can feel good to get your preface done quickly, you should write the rest of your paper first. Then, you’ll find it easy to create a clear overview.
• Include a strong quotation or story upfront. You want your paper to be full of substance. But that doesn’t mean it should feel boring or flat. Add a relevant quotation or surprising anecdote to the beginning of your introduction. This technique will pique the interest of your reader and leave them wanting more.
• Be concise. Research papers cover complex topics. To help your readers, try to write as clearly as possible. Use concise sentences. Check for confusing grammar or syntax . Read your introduction out loud to catch awkward phrases. Before you finish your paper, be sure to proofread, too. Mistakes can seem unprofessional.

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How to Write an Introduction For a Research Paper

Learn how to write a strong and efficient research paper introduction by following the suitable structure and avoiding typical errors.

An introduction to any type of paper is sometimes misunderstood as the beginning; yet, an introduction is actually intended to present your chosen subject to the audience in a way that makes it more appealing and leaves your readers thirsty for more information. After the title and abstract, your audience will read the introduction, thus it’s critical to get off to a solid start.  

This article includes instructions on how to write an introduction for a research paper that engages the reader in your research. You can produce a strong opening for your research paper if you stick to the format and a few basic principles.

What is An Introduction To a Research Paper?

An introduction is the opening section of a research paper and the section that a reader is likely to read first, in which the objective and goals of the subsequent writing are stated. 

The introduction serves numerous purposes. It provides context for your research, explains your topic and objectives, and provides an outline of the work. A solid introduction will establish the tone for the remainder of your paper, enticing readers to continue reading through the methodology, findings, and discussion. 

Even though introductions are generally presented at the beginning of a document, we must distinguish an introduction from the beginning of your research. An introduction, as the name implies, is supposed to introduce your subject without extending it. All relevant information and facts should be placed in the body and conclusion, not the introduction.

Structure Of An Introduction

Before explaining how to write an introduction for a research paper , it’s necessary to comprehend a structure that will make your introduction stronger and more straightforward.

A Good Hook

A hook is one of the most effective research introduction openers. A hook’s objective is to stimulate the reader’s interest to read the research paper.  There are various approaches you may take to generate a strong hook:  startling facts, a question, a brief overview, or even a quotation. 

Broad Overview

Following an excellent hook, you should present a wide overview of your major issue and some background information on your research. If you’re unsure about how to begin an essay introduction, the best approach is to offer a basic explanation of your topic before delving into specific issues. Simply said, you should begin with general information and then narrow it down to your relevant topics.

After offering some background information regarding your research’s main topic, go on to give readers a better understanding of what you’ll be covering throughout your research. In this section of your introduction, you should swiftly clarify your important topics in the sequence in which they will be addressed later, gradually introducing your thesis statement. You can use some  The following are some critical questions to address in this section of your introduction: Who? What? Where? When? How? And why is that?

Thesis Statement

The thesis statement, which must be stated in the beginning clause of your research since your entire research revolves around it, is the most important component of your research.

A thesis statement presents your audience with a quick overview of the research’s main assertion. In the body section of your work, your key argument is what you will expose or debate about it. An excellent thesis statement is usually very succinct, accurate, explicit, clear, and focused. Typically, your thesis should be at the conclusion of your introductory paragraph/section.

Tips for Writing a Strong Introduction

Aside from the good structure, here are a few tips to make your introduction strong and accurate:

• Keep in mind the aim of your research and make sure your introduction supports it.
• Use an appealing and relevant hook that catches the reader’s attention right away.
• Make it obvious to your readers what your stance is.
• Demonstrate your knowledge of your subject.
• Provide your readers with a road map to help them understand what you will address throughout the research.
• Be succinct – it is advised that your opening introduction consists of around 8-9 percent of the overall amount of words in your article (for example, 160 words for a 2000 words essay). 
• Make a strong and unambiguous thesis statement.
• Explain why the article is significant in 1-2 sentences.
• Remember to keep it interesting.

Mistakes to Avoid in Your Introduction

Check out what not to do and what to avoid now that you know the structure and how to write an introduction for a research paper .

• Lacking a feeling of direction or purpose.
• Giving out too much.
• Creating lengthy paragraphs.
• Excessive or insufficient background, literature, and theory.
• Including material that should be placed in the body and conclusion.
• Not writing enough or writing excessively.
• Using too many quotes.

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How to Write a Strong Introduction to a Research Paper

by Suzanne Davis | Jun 17, 2021 | Writing Essays and Papers , Writing Process

What makes an introduction powerful?

It’s an important question because an introduction is the first impression readers have of your research paper.  A strong introduction tells your readers not only what you will prove or show— it makes them want to read it .

You want to write an introduction that engages your reader, conveys the importance of your topic, and how and what you plan to demonstrate. These elements are what moves your audience, so they feel a desire to finish your research paper.

A powerful introduction says to the reader, “You must read me!”

3 Key Components of  Strong Introductions

So what are the key parts a good introduction should have? 1) a  compelling hook, 2) important background information and  3) a provable and specific thesis statement.  If you put those 3 pieces together you’ll have an effective introduction.

#1 Compelling Hook

A hook is the first 1 or 2 sentences of your paper.  It is meant to grab your readers’ attention, so they want to see what comes next.  If you want to learn about hooks check out my blog post, “7 Sensational Types of Essay Hooks.” https://www.academicwritingsuccess.com/7-sensational-types-of-essay-hooks/   There you’ll find more details about hooks and how to use them in your essays.

Here are 5 that work well in academic writing.

Powerful Statement /Declaration

With his hook, you begin with a sentence that makes a firm claim. For example, “Every day Facebook invades its users’ privacy.”  This sentence forces readers to reflect on whether or not they agree with this statement.  To do that they need to read your research paper.

In this case, most people have already formed an opinion about Facebook, and here you’ll either challenge or deepen their perspectives.

Fact/ Statistic

This hook is popular because people love to learn facts and statistics.  Studies can be very persuasive.  We regard studies as evidence or proof and place great value on statistics.  When readers see statistics, they want to know more about how it will relate to what you write.  However, make sure the facts you use come from a credible source.

Intriguing Question

When you start your paper or essay with a question, people will want to learn the answer to it.  They are naturally curious, so an intriguing question makes them want to continue reading. Make sure this question connects to what you will write about. An off-topic question is confusing.

Striking Quotation

Quotations are very popular. They can be inspiring and thought-provoking.  You can use quotations from famous people, experts, characters, or even people mentioned in your paper.  For example, if you are writing a case study you could select a quotation from one of the participants in the study. Quotations work well when they connect with the rest of your academic essay or paper. Make sure the relationship between the quotation and your writing is clear.

Simile/Metaphor 

In this hook, you take 2 different things and state how they are similar or the same. For example,  “Facebook is a digital version of prison people want to be in.” This sentence takes 2 different things “Facebook” and “prison” and claims they are alike. The comparison of Facebook being like a digital version of prison is a strong visual.  Your readers will want to see how you show a connection between these 2 things.

Both similes and metaphors accomplish the same thing in your hook. The difference is that a simile uses the words “like” and “as” to make the comparison, “Writing a novel is like running a marathon.”   A metaphor states one thing is another, “Writing a novel is running a marathon.”

#2 Important Background Information

The next group of sentences in your introduction express the circumstance and/or relevant information about your topic.  A lot of times you will see writers describe a problem, an issue or provide historical context.

Include the information people need to know to understand your topic and why it matters.  If you are writing about Facebook and privacy, people would want to see what the situation is and why privacy matters.  A good question to ask here is, “What is the context?” 

Also, start with general information first, and in the following sentences be more specific.  Those specific sentences lead to the most important piece of your introduction: your thesis statement.

#3 Provable and Specific Thesis Statement

A thesis statement is a sentence that describes what you will prove or show in your research paper. Think of it as the point of view (POV) or opinion you have about your topic.  It also guides how you will organize your essay or paper.

A strong thesis statement is clear and specific and one you can prove. For example, Children should not use digital devices until they are at least 3 years old because it lowers their attention span, limits social interaction, and causes sleep problems.

This thesis statement sets up the organization of your paper.  Each of the 3 effects of children using digital devices is part of the body. These sections of the body would go toward proving your thesis statement.

• Body Part A: Digital devices lower a child’s attention span.
• Body Part B:  Digital devices limit a child’s social interaction with others.
• Body Part C : Digital devices cause sleep problems.

So how do you create a thesis statement?

• Examine your topic, and briefly research it. I suggest reading any class notes you have and reading some background sources on your topic. What do people say about your topic?
• Ask yourself, “What do I think about this topic?”
• Brainstorm ideas and review them. What are the ideas that you can find research on?
• Write a sentence that connects your topic to what you will show, reveal, or prove about it . (Do not use the first person “I think” or “I feel” in your thesis statement).
• Evaluate your thesis statement. Is it specific? Can you prove it with evidence?

Write A Strong Introduction

There are different ways you can approach writing an introduction.  You could write the body of your paper first and then write the introduction.  You could write the thesis statement first and then write the hook and background information.

I like to write a thesis statement first, write the body of my paper, and then the conclusion. Afterward, I go back and write the rest of my introduction.

Some people like to begin with a hook and write a strong introduction before anything else.  Write the way that suits you best.  As long as you include a compelling hook, important background information, and a provable and specific thesis statement, you’ll have an impressive introduction.

Want help finding the perfect research paper topic? 

Sign-up below and get the free quick guide, Nail Your Research Paper Topic! 

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Set The Stage: How To Write A Research Paper Introduction

By Laura Brown on 31st March 2024

Are you planning to start with your research paper introduction? Your answer must be Yes! This is the reason you have landed on this page. By this time, you may also have completed your proposal. If, not, you may need a guide to write a research proposal .

But, if you are done with it and now looking forward to your research proposal, the first step would be to understand how to write an introduction of a research paper. Let’s not wait anymore and directly dig into the guide. We have prepared 9 simple steps with which you can master writing an introduction to a research paper!

Step 1: Provide An Overview

As you plan to comprehend the steps on how to write a research paper introduction, let’s kick things off by giving your readers a bird’s-eye view of your research.

Provide a brief overview of what your paper will cover and highlight the key topics and areas of focus. This sets the stage for what’s coming up and gives your readers a roadmap they can follow.

Social media has become an integral part of daily life for millions worldwide. Its pervasive influence extends beyond social interactions to various aspects of society, including mental health. This paper aims to explore the complex relationship between social media usage and mental well-being, shedding light on its multifaceted impact.

Step 2: Discuss The Significance

Next, you need to describe why your research matters. It is essential to discuss the significance of your topic. So, you need to highlight its relevance and importance in the broader context.

For this, you can explain why your readers should care about your research! Moreover, you should tell your readers how it contributes to existing knowledge or addresses a gap in the literature.

The significance of this research lies in the growing concern over the potential effects of excessive social media use on mental health. With the rise of social media platforms, concerns about increased stress, anxiety, and depression have emerged, prompting a need for comprehensive analysis and understanding.

Step 3: Identify Your Research Problems

Once you are done with significance, it’s time to pinpoint the specific problems or questions your research aims to address.

Here, you need to identify the challenges, gaps, or uncertainties in the current understanding of your topic that you are planning to resolve or explore. This part can be used to clarify the purpose of your study.

Despite the abundance of research on social media and mental health, gaps and inconsistencies persist. This study seeks to address key research problems, such as the nuanced effects of different social media platforms, the role of user behavior, and the influence of societal norms and perceptions.

Step 4: Outline The Objectives

To outline the objectives of your study, you should clearly state what you aim to achieve through your research. No matter if it’s to answer specific questions, test hypotheses, or provide insights into a particular phenomenon.

Remember that your objectives serve as guiding principles for your study and they will go on to shape the direction and focus of your research. If you feel like facing difficulty while identifying the objectives of your research, our research paper writing service has always got your back.

Our objectives are twofold: first, to examine the various ways in which social media impacts mental health, including both positive and negative effects; and second, to identify strategies for promoting mental well-being in the digital age.

Step 5: Define The Scope

The fifth step is to define the scope of your research. Now this is a critical step which will define where you can go as a researcher.

You should specify the boundaries and limitations of your study. Also, mention the specific aspects or variables you will focus on and those you will exclude. With this, you can define on how you will be managing your research.

Defining your scope also allows you to conduct a thorough investigation within the constraints of your resources and time frame.

This study focuses primarily on the psychological implications of social media use among young adults aged 18-30. We acknowledge that other demographic groups may experience unique challenges, but for the purpose of this paper, we will concentrate on this demographic due to its high social media engagement and susceptibility to mental health issues.

Step 6: Acknowledge Limitations

It’s essential to acknowledge any potential limitations or constraints that may affect your research. You should always be transparent about factors such as time, resources, or access to data that could impact the scope or outcomes of your study.

Think about it in depth and check out for the lack of resources that can really impact your research!

By acknowledging these limitations upfront, you can demonstrate a realistic and honest approach to your research. It will pave the way for highlighting opportunities for future inquiry or refinement.

It’s important to recognise the limitations of this study, including the reliance on self-reported data, the potential for selection bias, and the dynamic nature of social media platforms. These limitations may impact the generalisability of our findings and should be considered in interpreting the results.

Step 7: Propose Methodology

As you plan on how to write an intro for a research paper, methodology stands tall at the seventh step. Outline the methodology you plan to use to conduct your research, be it primary or secondary research . You need to discuss the specific techniques, procedures, or approaches you are going to employ to gather and analyse data.

Whether your methodology involves qualitative interviews, quantitative surveys, or experimental design, provide a rationale for your chosen approach and explain how it aligns with your research objectives.

Our methodology will involve a mixed-methods approach, combining quantitative surveys to assess social media usage patterns and mental health outcomes with qualitative interviews to explore individual experiences and perceptions. This approach allows for a comprehensive understanding of the complex interplay between social media and mental well-being.

Step 8: Present Your Thesis Statement

Now finally it’s time to introduce your thesis statement, which succinctly summarises the main argument or central claim of your research paper.

Make sure that you are writing a clear, concise, and debatable thesis statement. Do not forget to mention the key insights or conclusions you intend to support throughout your paper.

You may ask why is it necessary to provide a thesis statement so early. It is essential so that you can provide readers with a roadmap for understanding the overarching purpose and focus of your research.

This paper argues that while social media can have both positive and negative impacts on mental health, its overall effect depends on various factors, including individual differences, usage patterns, and platform design. By examining these factors, we aim to provide insights into how social media can be leveraged to promote mental well-being in today’s digital society.

Step 9: Describe The Structure

Finally, the last step on how to make an introduction in a research paper involves describing the structure of your research paper. You need to provide an overview of the structure of your research paper.

• Briefly outline the main sections or chapters of your paper
• Explain how they contribute to your overall argument or analysis.
• Consider including subsections or key points within each section
• Give your readers a preview of the content that is coming.

Remember that it is a crucial step! Describing the structure of your paper helps your readers navigate through the document and understand the logical progression of your ideas.

The paper is structured as follows: first, we will review the existing literature on social media and mental health to provide context for our study. Next, we will present our methodology and findings, followed by a discussion of the implications for research and practice. Finally, we will conclude with recommendations for future research and interventions.

Concluding On How To Write A Research Paper Introduction

Coming up with an introduction for your research paper is a simple task! Students often consider it a challenging one, but if you are able to divide it into smaller chunks, you will be able to attempt it smoothly.

We have divided the whole process of how to write an introduction in a research paper into 9 simple steps. This is our take on how students should proceed with it. What’s your say? Did you find this post valuable? Do not forget to share your views with us.

Let Us Answer Your Queries

How long should an introduction be in a research paper.

The introduction is typically 10% of your complete research. You need to keep it short. For a research paper of around 10,000 words, you need to write a 1000-word introduction.

How do I write an introduction for a research paper?

Writing an introduction to your research paper is quite simple. All you need is to begin with background information, state the research problem, and highlight the significance. Then present the thesis statement, and outline the paper’s structure in a clear and engaging manner.

How to write a good introduction for a research paper?

If you are willing to write a good introduction for your research paper, follow these 3 tips. – State your research problem clearly. – Start your intro with an engaging hook-up line. – Demonstrate the significance of your research to your field of study.

Laura Brown, a senior content writer who writes actionable blogs at Crowd Writer.

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• A Research Guide
• Research Paper Guide

How to Write an Introduction for a Research Paper

• Purpose of intro
• Key elements
• Writing an effective intro
• Step-by-step guide
• Research intro checklist
• Introduction formats
• Good and bad examples

An introductory paragraph is vital for any academic paper. It allows you to show reviewers why your research topic is worth reading about. In this article, we will explore the tips to make a good introduction paragraph. You’ll get a step-by-step tutorial on writing your paper’s informative yet laconic intro.

What is the purpose of an introduction?

The purpose of a research paper intro is to provide an overview and context for the study being conducted. A research paper engages the reader, establishes the importance of the research topic, and outlines the study’s objectives and scope.

The paper intro also presents the question or hypothesis and summarizes relevant background characteristics and existing literature.

An effective introduction helps the reader understand the significance and relevance of the research paper and sets the stage for the subsequent sections. The introduction captures the reader’s attention and creates a foundation for understanding the research and its contributions.

The key elements of a scientific paper introduction

The introduction of your research paper should include several key elements, including the problem statement, hypothesis/thesis/research question, purpose, and background.

Let’s explore each of these parts of the research paper intro in detail:

• Problem Statement : identifies the specific issue or gap in knowledge that the research paper aims to address. It highlights the problem’s relevance, significance, and potential impact on the field of study. The problem statement sets the stage for the research by clearly stating the project or research gap.
• Hypothesis / Thesis / Research Question : a paper hypothesis predicts the relationship between variables, a thesis statement presents the main argument or claim, and a research question seeks to put a specific aspect on a research paper.
• Purpose: describes the overall objective or goal the research paper aims to achieve. It outlines the researcher’s intention and provides a clear direction for the investigation. The purpose statement typically explains why the research is being conducted and what the researcher hopes to accomplish by the end of the study.
• Background : provides the necessary context and information to familiarize readers with the research paper. It presents a concise review of the relevant literature, previous studies, and theoretical frameworks that have shaped the understanding of the problem.

Shortly, the introduction section of a research paper combines these key elements to introduce the problem, state the hypothesis/thesis/research question, define the paper’s purpose, and provide the background necessary for readers to understand the significance and context of the study.

How to write an effective intro?

To start an introduction for a research paper, consider the following steps:

• Hook the reader : begin with a compelling opening sentence or a thought-provoking statement that grabs the reader’s attention. This could be an interesting fact, a relevant anecdote, or a surprising statistic related to your research paper.
• Provide background information : offer a brief overview of the paper and its significance in the field. This helps to improve the structure of an introduction and demonstrate why it is important to investigate the point further in a paper.
• State the problem : clearly articulate the problem statement or research gap your study aims to address. Explain the specific issue or gap in knowledge that your research paper seeks to explore, emphasizing its relevance and potential impact.
• Present the research question/hypothesis/thesis : formulate a concise and focused research question, hypothesis, or thesis statement in the intro that guides your scientific paper. This sets the direction for your research and provides a clear focus for the reader.
• Outline the purpose and objectives : explain the overall purpose of your research paper and the specific objectives you aim to achieve. This helps readers understand why your study is being conducted and what you hope to accomplish.
• Preview the structure : briefly introduce the organization and structure of your research paper. Mention the main sections or components that will be covered, giving readers a sense of what to expect as they continue reading the paper.

Remember, the intro should be concise and engaging, providing a clear roadmap for your research and capturing the reader’s interest from the very beginning. There are different ways to start a research paper, and you can pick the intro that suits you best.

Writing an introduction to a research paper: key steps

Here’s a short guide on getting you started with an introduction:

• Start with an attention-grabbing opening : begin your intro with a captivating statement, a relevant quote, a surprising fact, or an intriguing anecdote. This will engage the reader’s interest and make them curious about your research paper.
• Provide background information : write a brief overview of the research topic to provide context and establish the importance of the subject matter. Discuss key concepts, definitions, or historical background relevant to your study. This section should help the reader understand the broader context of your research paper.
• State the research problem or gap : clearly define the specific problem or research gap your study aims to address. Explain why this problem is significant and deserving of investigation. This helps the reader understand the purpose and relevance of your research paper.
• Present your research question or thesis statement : formulate a clear and concise research question, hypothesis, or thesis statement that serves as the central focus of your study. This statement should guide your research paper and articulate your introduction format.
• Outline the structure of the paper : write a brief preview of your research paper’s main sections and organization. This helps the reader understand the flow of your paper and what to expect in each section. Provide a roadmap by mentioning the key points or arguments discussed in subsequent sections.

By following these steps, you can create an introduction that grabs the reader’s attention and sets the stage for the rest of your research paper, clearly understanding your study’s problem, purpose, and structure.

Writing a checklist for a proper college paper introduction

Here’s a short writing checklist for a research paper intro:

• Attention-grabbing opening:
• Does the research paper introduction example start with a compelling statement, relevant quote, surprising fact, or intriguing anecdote?
• Is the opening engaging enough to capture readers’ attention and make them curious about the research paper?
• Background information:
• Have you provided a concise overview of the research topic, including relevant definitions, concepts, or historical context?
• Does the background information help the reader understand the broader context and importance of the subject matter?
• Clear problem statement:
• Have you clearly stated the specific problem or research gap that your study aims to address?
• Does a research introduction have a well-defined, strong, and significant problem statement?
• Research question or thesis statement:
• Have you presented a clear and concise research question, hypothesis, or thesis statement that guides your paper?
• Does the research question or thesis statement align with the problem statement and set the direction for your research paper?
• Structure and organization:
• Did you write a brief overview of the structure and organization of the research paper?
• Does the introduction outline the main sections or components covered in the paper?
• Coherence and flow:
• Is the intro logically organized? Does it have smooth transitions between ideas and paragraphs?
• Does the intro flow smoothly from the opening to the problem statement, research question, and purpose?
• Conciseness and clarity:
• Have you kept the introduction concise, avoiding unnecessary details or tangents?
• Is the language clear, avoiding jargon or overly technical terms that may confuse the reader?
• Relevance and significance:
• Have you clearly explained the relevance and significance of the research topic and the paper’s potential impact?
• Does the introduction effectively communicate why your research is important and worth exploring?

This checklist will help you to review your research essay introduction to ensure it effectively grabs the reader’s attention, provides necessary background information, states the problem clearly, presents a focused research question or thesis statement, outlines the structure of the paper, and maintains coherence and clarity throughout.

Types of intro formats

Different academic disciplines may follow specific formatting styles for research introduction, such as MLA (Modern Language Association), APA (American Psychological Association), Chicago, ASA (American Sociological Association), and AMA (American Medical Association).

To write an introduction paragraph, you should understand the differences between the most common academic formats for your future paper.

MLA (Modern Language Association):

• Primarily used in humanities, literature, and arts disciplines.
• Features in-text citations using the author-page format (e.g., “Smith 45”).

APA (American Psychological Association):

• Commonly used in social sciences, psychology, and education.
• Utilizes in-text citations with the author-date format (e.g., “Smith, 2019”).
• Often used in history, humanities, and some social sciences.
• Offers two styles: the notes-bibliography system and the author-date system.
• Includes a bibliography page to list all sources used.

ASA (American Sociological Association):

• Primarily used in sociology and related social sciences.
• Utilizes in-text citations with the author-date format (e.g., “Smith 2019”).

AMA (American Medical Association):

• Commonly used in medical, health, and biological sciences.
• Features in-text citations with a superscript number (e.g., “Smith^1”).
• Emphasizes accuracy and consistency in citation style.

All formatting styles mean a set of rules and guidelines for citing sources, formatting headings, page layout, and referencing. It’s important to consult the specific style guide or manual associated with your field of study before you write.

These might include guidelines provided by your institution to ensure proper paper formatting and adherence of a research introduction to the chosen style.

Research introduction sample

Now that you know how the idea goes in the introduction of a research paper, let’s see the practical examples of good and bad introductions and discuss their differences.

Good example:

Title: “Exploring the Impact of Climate Change on Biodiversity: A Comprehensive Analysis”

Introduction:

Climate change is a pressing global issue that has far-reaching consequences for our planet. Its effects on various ecosystems, particularly biodiversity loss, have attracted significant attention from researchers and policymakers alike.

This research paper aims to provide a comprehensive analysis of the impact of climate change on biodiversity, focusing on key regions and species vulnerable to these changes. By examining the latest scientific literature, empirical studies, and expert opinions, we will explore the complex interplay between climate change and biodiversity loss, shed light on the underlying mechanisms, and propose potential mitigation strategies.

Understanding these dynamics is crucial for developing effective conservation strategies and promoting sustainable practices that will help preserve our planet’s invaluable natural heritage.

Bad example:

Title: “Climate Change and Biodiversity”

Climate change and biodiversity are two important topics that have received considerable attention recently. Climate change refers to the long-term alteration of temperature and precipitation patterns, while biodiversity encompasses the variety of life forms found on Earth.

In this research paper, we will discuss the impact of climate change on biodiversity and explore various examples and case studies. The paper will also highlight the significance of addressing this issue and present potential solutions.

By delving into this subject, we aim to contribute to the existing body of knowledge and raise awareness about the importance of protecting biodiversity in climate change.

To begin an introduction paragraph, don’t provide too much background or theory at once. Remember to arrange your thoughts concisely while keeping the important information for the paper body.

A good intro should answer the four basic questions:

• What was I studying?
• Why was this topic important to investigate?
• What did we know about this topic before I did this study?
• How will this study advance our knowledge?

Remember that you might not get a second chance to create a positive first impression. That’s why it’s equally important to keep your paper laconic and to end an introduction paragraph with a call to action to read your research paper.

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What is a "good" introduction?

Citing sources in the introduction, "introduction checklist" from: how to write a good scientific paper. chris a. mack. spie. 2018..

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This is where you describe briefly and clearly why you are writing the paper. The introduction supplies sufficient background information for the reader to understand and evaluate the experiment you did. It also supplies a rationale for the study.

• Present the problem and the proposed solution
• Presents nature and scope of the problem investigated
• Reviews the pertinent literature to orient the reader
• States the method of the experiment
• State the principle results of the experiment

It is important to cite sources in the introduction section of your paper as evidence of the claims you are making. There are ways of citing sources in the text so that the reader can find the full reference in the literature cited section at the end of the paper, yet the flow of the reading is not badly interrupted. Below are some example of how this can be done:     "Smith (1983) found that N-fixing plants could be infected by several different species of Rhizobium."     "Walnut trees are known to be allelopathic (Smith 1949,  Bond et al. 1955, Jones and Green 1963)."     "Although the presence of Rhizobium normally increases the growth of legumes (Nguyen 1987), the opposite effect has been observed (Washington 1999)." Note that articles by one or two authors are always cited in the text using their last names. However, if there are more than two authors, the last name of the 1st author is given followed by the abbreviation et al. which is Latin for "and others". 

From:  https://writingcenter.gmu.edu/guides/imrad-reports-introductions

• Indicate the field of the work, why this field is important, and what has already been done (with proper citations).
• Indicate a gap, raise a research question, or challenge prior work in this territory.
• Outline the purpose and announce the present research, clearly indicating what is novel and why it is significant.
• Avoid: repeating the abstract; providing unnecessary background information; exaggerating the importance of the work; claiming novelty without a proper literature search. 
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• Published: 28 May 2024

Gut microbiome remodeling and metabolomic profile improves in response to protein pacing with intermittent fasting versus continuous caloric restriction

• Alex E. Mohr   ORCID: orcid.org/0000-0001-5401-3702 1 , 2 ,
• Karen L. Sweazea 1 , 2 , 3 ,
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• Paniz Jasbi 4 , 5 ,
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• Karen M. Arciero 7 ,
• Eric Gumpricht 8 &
• Paul J. Arciero   ORCID: orcid.org/0000-0001-7445-6164 7 , 9  

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The gut microbiome (GM) modulates body weight/composition and gastrointestinal functioning; therefore, approaches targeting resident gut microbes have attracted considerable interest. Intermittent fasting (IF) and protein pacing (P) regimens are effective in facilitating weight loss (WL) and enhancing body composition. However, the interrelationships between IF- and P-induced WL and the GM are unknown. The current randomized controlled study describes distinct fecal microbial and plasma metabolomic signatures between combined IF-P ( n  = 21) versus a heart-healthy, calorie-restricted (CR, n  = 20) diet matched for overall energy intake in free-living human participants (women = 27; men = 14) with overweight/obesity for 8 weeks. Gut symptomatology improves and abundance of Christensenellaceae microbes and circulating cytokines and amino acid metabolites favoring fat oxidation increase with IF-P (p < 0.05), whereas metabolites associated with a longevity-related metabolic pathway increase with CR (p < 0.05). Differences indicate GM and metabolomic factors play a role in WL maintenance and body composition. This novel work provides insight into the GM and metabolomic profile of participants following an IF-P or CR diet and highlights important differences in microbial assembly associated with WL and body composition responsiveness. These data may inform future GM-focused precision nutrition recommendations using larger sample sizes of longer duration. Trial registration, March 6, 2020 (ClinicalTrials.gov as NCT04327141), based on a previous randomized intervention trial.

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Introduction.

As a principal modulator of the gut microbiome (GM) and weight status, nutritional input holds great therapeutic promise for addressing a wide range of metabolic dysregulation 1 . Dependent on the host for nutrients and fluid, one of the main processes by which the GM affects host physiology is producing bioactive metabolites from the gastrointestinal (GI) contents. Nutrient composition, feeding frequency, and meal timing impact this dependency 2 , 3 . To maintain a stable community and ecosystem, the GM must regulate its growth rate and diversity in response to nutrient availability and population density 4 . Such maintenance is affected by caloric restriction (CR) coupled with periods of feeding and intermittent fasting (IF) 5 . Moreover, we’ve recently shown the nutritional composition and meal frequency during these periods alter the metabolizable energy for the host 6 . The current study incorporates protein pacing (P), defined as four meals/day consumed evenly spaced every 4 h, consisting of 25–50 g of protein/meal 7 , 8 , 9 . Indeed, we have previously characterized a dietary approach of calorie-restricted IF-P combined and P alone 7 , 8 . These studies included nutrient-dense meal replacement shakes, along with whole foods, to quantitatively examine beneficial changes in body composition and cardiometabolic, inflammatory, and toxin-related outcomes in healthy and overweight individuals 7 , 8 , 10 , 11 , 12 . Further, recent preclinical work in mice has identified dietary protein as having anti-obesity effects after CR that are partially modulated through the GM 13 . Thus, the need to examine this in humans is warranted.

In this current work, we compare the effects of two low-calorie dietary interventions matched for weekly energy intake and expenditure; continuous caloric restriction on a heart-healthy diet (CR) aligned with current United States (US) dietary recommendations 14 versus our calorie-restricted IF-P diet 8 , 15 , in forty-one individuals with overweight or obesity, over an 8-week intervention. We hypothesize an IF-P diet may favorably influence the GM and metabolome to a greater extent than a calorie-matched CR alone. This exploratory investigation utilizes data and samples from a randomized controlled trial (NCT04327141) that compares the effects of the CR versus IF-P diet on anthropometric and cardiometabolic outcomes, as previously published 15 . As an additional analysis, we select “high” and “low” responders based on relative weight loss (WL) for a subgroup examination of the IF-P diet to better elucidate potential differential responses to intermittent fasting and protein pacing. Of special note, one individual lost 15% of their initial body weight over the 8-week intervention; this individual is followed longitudinally for a year to explore the dynamics of their GM and fecal metabolome. Novel findings from the current study shows an IF-P regimen results in improved gut symptomatology, a more pronounced community shift, and greater divergence of the gut microbiome, including microbial families and genera, such as Christensenellaceae , Rikenellaceae , and Marvinbryantia , associated with favorable metabolic profiles, compared to CR. Furthermore, IF-P significantly increases cytokines linked to lipolysis, weight loss, inflammation, and immune response. These findings shed light on the differential effects of IF-P as a promising dietary intervention for obesity management and microbiotic and metabolic health.

Intermittent fasting - protein pacing (IF-P) significantly influences gut microbiome (GM) dynamics compared to calorie restriction (CR)

We compared an IF-P vs. a CR per-protocol dietary intervention (matched for total energy intake and expenditure) over eight weeks to compare changes in weight, cardiometabolic outcomes, and the GM in men and women with overweight/obesity (IF-P: n  = 21; CR: n  = 20). One participant in each group were lost to follow-up due to non-compliance with dietary intervention (Fig.  1a ; CONSORT flow diagram: Supplementary Fig.  S1a ). The primary outcomes of dietary intake, body weight and composition responses, cardiometabolic outcomes, and hunger ratings after both dietary interventions are provided in our companion paper 15 . Briefly, after a one-week run-in period consuming their usual dietary intake (baseline diet), with no differences between groups at baseline for any dietary intake variable 15 , both dietary interventions significantly reduced total fat, carbohydrate, sodium, sugar, and energy intake by approximately 40% (~1000 kcals/day) from baseline levels (Fig.  1b ; Supplementary Data  1 ). By design, IF-P increased protein intake greater than CR during the intervention. The IF-P regimen consisted of 35% carbohydrate, 30% fat, and 35% protein for five to six days per week and a weekly extended modified fasting period (36–60 h) consisting of 350–550 kcals per day using randomization, as detailed previously 7 , 8 , 9 , 10 , 15 . In comparison, the CR regimen consisted of 41% carbohydrate, 38% fat, and 21% protein in accordance with current US dietary recommendations (Supplementary Table  S1 ) 14 , 16 . Using two-way factorial mixed model analysis of variance (ANOVA), significant macronutrient decreases drove energy reduction from dietary fat and carbohydrate ( p  < 0.001), with increased protein in the IF-P compared to CR ( p  < 0.001; Supplementary Fig.  S1b ; Supplementary Data  1 ). Regarding GI functioning and GM modulation, IF-P significantly decreased sugar and increased dietary fiber relative to CR (IF-P; pre, 20 ± 2 vs. post, 26 ± 2: CR; pre, 24 ± 3 vs. 24 ± 2 g/day; p  < 0.05). Despite similar average weekly energy intake (~9000 kcals/week) and physical activity energy expenditure (~350 kcals/day; p  = 0.260) during the intervention, participants following the IF-P regimen lost significantly more body weight (−8.81 ± 0.71% vs. −5.40 ± 0.67%; p  = 0.003; Fig.  1c ; Supplementary Data  1 ) and total, abdominal, and visceral fat mass and increased fat-free mass percentage (~2×; p  ≤ 0.030), as previously reported 15 . In addition, within-group analyses revealed a significant decrease in the reported frequency of total and lower-moderate GI symptoms (GI symptom rating score [GSRS] ≥4) over time for both IF-P and CR participants. However, when comparing the two dietary interventions at each time point, a more substantial reduction was observed in IF-P participants compared to CR participants (i.e., −9.3% vs. −5.4% and −13.2% vs. −3.9%, respectively; Table  1 ). The increased protein and lower sugar intake in IF-P compared to CR may have favorably mediated the GM and symptomatology.

a Study design with baseline participant characteristics. A registered dietitian counseled individuals from both groups each week. Time points with data collection are shown for both IF-P and CR participants. Icons created using BioRender.com. b Total daily caloric intake at each time point was not significantly different between IF-P and CR diet groups (two-sided Student’s t -test, p  < 0.05). Adjusted values are displayed by dividing total weekly intake by seven, to account for the fasting periods of IF-P. c IF-P participants lost significantly more weight over time versus CR participants. Points connected by line represent percent of weight compared to baseline weight for each participant. d Overall gut microbial colonization, as demonstrated by qPCR-based quantification of 16S rRNA gene copies per gram wet weight was unaffected by time or intervention (linear-mixed effects [LME] model, two-sided p  > 0.05). Alpha diversity metrics, e observed amplicon sequence variants (ASVs), and f Phylogenetic diversity at the ASV level significantly increased over time, independent of the intervention. g Intra-individual changes in GM community structure from baseline to weeks four and eight in IF-P participants shifted significantly throughout the IF-P intervention compared to CR as measured by the Bray-Curtis dissimilarity index (two-sided Wilcoxon rank-sum test). All box and whiskers plots display the box ranging from the first to the third quartile, and the center the median value, while the whiskers extend from each quartile to the minimum or maximum values. Heatmap of significant changes in h family- and i genus-level bacter i a by intervention. Colors indicate the within-group change beta coefficients over time for each cell, and asterisks denote significance. Black-white annotations on the bottom denote the significance of between-group change difference (by MaAsLin2 group × time interactions; p -values were corrected to produce adjusted values [ p .adj] using the Benjamini–Hochberg method). For all panels, IF-P: n  = 20, CR: n  = 19. Source data are provided as a Source Data file.

The substantial reduction in calorie intake of both groups (~40% from baseline) led us to investigate its potential impact on transient microbial colonization in the gut, as estimated by 16S rRNA gene copies (linear-mixed effects model [LME] time effect, p  = 0.114; Fig.  1d ; Supplementary Data  2 ). While it might be expected that a significant reduction in calorie intake could influence gut microbial colonization, our findings indicate that this reduction did not reach statistical significance within the timeframe of our study. This result contrasts with previous research that imposed more substantial energy restriction, such as a four-week regimen of ~800 kcal/day in participants with overweight/obesity, where overall gut microbial colonization notably decreased 4 . In addition to assessing microbial colonization, we also investigated whether the calorie reduction significantly influenced principal stool characteristics, including wet stool weight, Bristol stool scale (BSS), and fecal pH ( p  ≥ 0.066; Table  1 ). However, we did not observe statistically significant changes in these parameters over the course of the study. Moreover, there were no significant differences between the two dietary intervention groups over time (interaction effect, p  ≥ 0.051). In contrast, there were significant time effects for observed amplicon sequence variants (ASVs) and phylogenetic diversity (LME time effect, p  ≤ 0.023; Fig.  1e, f ; Supplementary Data  2 ), with values increasing at weeks four and eight compared to baseline (pairwise comparisons, p  ≤ 0.048); however, no interaction was observed for either alpha diversity metric (group × time effect, p  ≥ 0.925). To rule out the potential confounding effects of GI transit time 17 , BSS (as a surrogate marker) and stool pH were not significantly correlated with alpha diversity (Spearman correlations, p  ≥ 0.210). In relation to community composition, much of the intervention variance could be attributed to individual response upon testing nested permutational analysis of variance (PERMANOVA; R 2  = 0.749, p  = 0.001; Supplementary Table  S2 ), showcasing the highly individualistic landscape of the human GM in response to dietary intervention. However, a significant 1.8% of the variance was accounted for by the group × time interaction ( p  = 0.001). Moreover, individual responses over time showed variance between the two dietary interventions (PERMANOVA, R 2  = 0.123, p  = 0.003). This variability was apparent by assessing intra-individual differences, where a pronounced increase in Bray-Curtis dissimilarity was observed in the IF-P compared to the CR group after four (median Bray-Curtis dissimilarity, 0.53 [IQR: 0.47–0.61] vs. 0.38 [IQR: 0.33–0.47]) and eight weeks (0.50 [IQR: 0.41–0.55] vs. 0.39 [IQR: 0.33–0.45]; Fig.  1g ; Wilcoxon rank-sum test, p  ≤ 0.005).

To understand the taxa driving this GM variation from baseline to weeks four and eight between the two dietary interventions, we constructed MaAsLin2 linear-mixed models with the individual participant as a random factor 18 . We observed differential abundance patterns at the family and genus level in response to the IF-P but not the CR intervention. Of the 28 family and 69 genus-level features captured after filtering, a respective total of six and 18 taxa displayed significant interaction effects, with all significant time effects occurring from IF-P ( p .adj ≤ 0.10; Fig. 1h, i ; Supplementary Data  3 , 4 ). Notably, the changes observed at the four-week mark were more pronounced compared to those at eight weeks. These early alterations may signify an initial adaptation phase during which microbial populations respond to the modified substrate availability and nutrient composition, suggesting a degree of community resilience 19 . Increases were sustained to the third fecal collection for the family Christensenellaceae and the genera Incertae Sedis ( Ruminococcaceae family), Christensenellaceae R-7 group , and UBA1819 ( Ruminococcaceae family) (effect size > 2.0). Christensenellaceae is well regarded as a marker of a lean (anti-obesity) phenotype 20 and is associated with higher protein intake 21 . Other notable increases included Rikenellaceae , which, like Christensenellaceae , has been linked to reduced visceral adipose tissue and healthy metabolic profiles 22 , and Marvinbryantia , a candidate marker for predicting long-term weight loss success in individuals with obesity 23 . In addition, IF-P increased Ruminococcaceae , which has been noted to have an increased proteolytic and lipolytic capacity 24 . This shift in IF-P participants likely represents a change in GM substrate fermentation preferences as the diet regimen (relative protein and carbohydrate) and energy restriction is expected to increase the proteolytic: saccharolytic potential ratio 25 . In contrast, all taxa that decreased in IF-P participants were butyrate producers. These included the family Butyricicoccaceae and several genera such as Butyricicoccus (week four), Eubacterium ventriosum group (weeks four and eight), and Agathobacter (week four) (effect size < −2.0). When comparing monozygotic twin pairs, Eubacterium ventriosum group and another reduced genus, Roseburia , were more abundant in the higher body mass index (BMI) siblings 26 . Others, such as the mucosa-associated Butyricicoccus and Erysipelotricaceae UCG-003, have been positively correlated with insulin resistance and speculated to contribute to impaired glycolipid metabolism 27 .

Despite these changes in GM composition and increased fiber intake (+30% vs. baseline) of the IF-P participants 15 , we did not detect a significant shift in the abundance of the principal fecal short-chain fatty acids (SCFAs), acetate, propionate, butyrate, or valerate, as assessed by gas chromatography-mass spectrometry (GC–MS) (LME, p  ≥ 0.470; Supplementary Fig.  S1c ; Supplementary Data  5 ). Several factors likely contribute to this finding. For example, the distinct physical-chemical properties of fiber sources between IF-P and CR are inherently different. Participants adhering to the IF-P diet consumed most of their dietary fiber as liquid meal replacements (shakes) that are rich in non-digestible, oligosaccharide dietary-resistant starch 5 (RS5). In contrast, subjects on the CR regimen consumed their fiber from whole food sources such as vegetables, whole grains, and legumes. These fiber sources provided a mixture of soluble and insoluble fibers and a more complex fiber profile than IF-P participants. Moreover, even similar fiber profiles may function differently due to differences in food matrices and/or food preparation (cooking, raw consumption, etc.). Also of relevance is the timing of their fiber consumption. IF-P participants’ fiber intake was concentrated in fiber-rich shakes, offering immediate availability of fiber to the GI tract. In contrast, CR participants consumed fiber through whole foods, leading to a slower digestion and absorption process influenced by individual digestive transit times and enzymatic profiles. Interestingly, our results parallel recent work where participants more than doubled their fiber intake without affecting fecal SCFAs 28 . The disparate findings may be due to the type of dietary-resistant starch (RS) as a component of the nutrition regimen. In the current study, RS5 was included in the meal replacement shakes (eight grams/shake, two shakes/day, 16 g/day total). Prior research supports resistant starch intakes of >20 g/day favorably modulate SCFA production, primarily butyrate, over four to 12-week interventions 29 , 30 . Moreover, this lack of response in fecal SCFAs in both groups may have been further compounded by the significant reduction in energy intake in both groups, where the epithelia of the GI tract may have absorbed any potential increase in SCFAs from the dietary shift. It is worth noting that stool analysis may not be the most reliable biological surrogate for capturing SCFA flux over time 28 . Nevertheless, the changes in nutrient quality, timing, ratios, and the observed shift toward proteolytic activity suggest that the luminal matrix of digesta in the IF-P group impacted substrate availability for GM. This effect appears to be an influencing force in driving the observed beneficial shifts in microbial communities, such as Christensenellaceae and Incertae Sedis , as well as improvements in GI symptomatology in IF-P compared to CR. These results underscore the complexity of dietary influences on GM and highlight the need for further research to explore the impact of liquid meal replacements versus whole food sources on GM changes and SCFA status.

IF-P modulates circulating cytokines and gut microbiome taxa compared to CR

Caloric restriction and WL have been well known to positively influence inflammatory cytokine expression, with GM now emerging as an important modulator 31 . Surveying a panel of 14 plasma cytokines, we noted significant interaction (group × time) effects for IL-4, IL-6, IL-8, and IL-13 (LME, p  ≤ 0.034; Fig.  2a–d ; Supplementary Table  S3 ; Supplementary Data  6 ). These cytokines exhibited increases at weeks four and/or eight compared to baseline exclusively in the IF-P group (pairwise comparisons, p .adj ≤ 0.098), while no significant changes were observed in the CR group ( p .adj ≥ 0.562). Notably, IL-4 has been reported to display lipolytic effects 32 , and IL-8 has been positively associated with weight loss and maintenance 33 . Regarded as a proinflammatory myokine, IL-6 can acutely increase lipid mobilization in adipose tissue under fasting or exercise conditions 34 , 35 , 36 . IL-13 may be important for gut mucosal immune responses and is a stimulator of mucus production from goblet cells 37 , which has been recently reported to be influenced during a two-day-a-week fasting regimen in mice 38 . These results were of note considering the significant total body weight, fat, and visceral fat loss in the IF-P compared to the CR group. Surprisingly, correlational analysis with change (post – pre) in anthropometric and select plasma biomarker values with the cytokine profile did not reveal any significant associations after correcting for multiple testing effects ( p .adj ≥ 0.476; Supplementary Data  7 ). Plasma cytokines were, however, correlated with microbial composition for samples collected in the IF-P group during the intervention period (weeks four and eight) using graph-guided fused least absolute shrinkage and selection operator (GFLASSO) regression, revealing associations between cytokine-taxa pairs (Supplementary Fig.  S2a ). Of the four cytokines that increased in IF-P participants, we identified multiple significant correlations: Colidextribacter (rho = −0.55, p .adj = 0.015), Ruminococcus gauvreauii group (rho = 0.50, p .adj = 0.036), and Intestinibacter (rho = 0.45, p .adj = 0.086) with IL-4 (Supplementary Fig.  S2b ) and an unclassified genus from Oscillospiraceae (rho = −0.53, p .adj = 0.019), Colidextribacter (rho = −0.52, p .adj = 0.019), and Ruminoccus gauvreauii group (rho = 0.51, p .adj = 0.019) with IL-13 (Supplementary Fig.  S2c ).

a IL-4, b IL-6, c IL-8, and d IL-13: Each panel shows the cytokine concentration levels. Significant time effects and interaction effects (group × time) were detected using linear-mixed effects models (LME, two-sided p  < 0.05), indicating differential changes over the intervention period. IF-P participants exhibited significant increases in cytokine levels compared to baseline, as evidenced by pairwise comparisons adjusted for multiple testing using the Benjamini–Hochberg method (two-sided p .adj < 0.10). All box and whiskers plots display the box ranging from the first to the third quartile, and the center the median value, while the whiskers extend from each quartile to the minimum or maximum values. For all panels, IF-P: n  = 20, CR: n  = 19. Source data are provided as a Source Data file.

Displaying negative correlations for IL-4 and IL-13, Colidextribacter has been shown to be positively correlated to fat accumulation, insulin, and triglyceride levels in mice fed a high-fat diet 39 and positively correlated with products of lipid peroxidation, suggesting its potential role in promoting oxidative stress 40 . Conversely, Ruminoccus gauvreauii group was positively correlated with IL-4 and IL-13. Although limited information is available regarding the host interactions of this microbe, this genus is considered a commensal part of the core human GM and able to convert complex polysaccharides into a variety of nutrients for their hosts 41 . While these findings highlight the potential interplay between specific microbes and cytokine profiles, the directional influence—whether microbial changes drive cytokine alterations or vice versa—cannot be determined in this study setting. Furthermore, despite the change in cytokine profiles in the IF-P group, we did not detect any significant time or group × time effects when measuring lipopolysaccharide-binding protein (LBP; Δ pre/post, IF-P: 0.24 ± 0.31 vs CR: −0.93 ± 0.49 μg/mL; p  ≥ 0.254), a surrogate marker for gut permeability 42 . While the GM plays a crucial role in modulating the gut-immune axis, the observed cytokine fluctuations and microbial associations might also involve other factors. These include the production of specific metabolites due to shifts in microbial composition as well as the influence of the dietary regimen itself, which may have a central role in shaping these interactions.

IF-P and CR yield distinct circulating metabolite signatures and convergence of multiple metabolic pathways

To understand the potential differential impact of IF-P versus CR on the host, we surveyed the plasma metabolome, reliably detecting 136 plasma metabolites across 117 samples (i.e., QC CV < 20% and relative abundance > 1000 in 80% of samples). Based on outlier examination (random forest [RF] and principal component analysis [PCA]), no samples were categorized as outliers, and all data were retained for subsequent analysis. Metabolomic profile shifts were observed in both IF-P and CR groups compared with baseline (Canberra distance), however, these did not differ significantly by group or time (weeks four and eight; Wilcoxon rank-sum test, p  ≥ 0.087; Supplementary Fig.  S3a ). We prepared a general linear model (GLM) with age, sex, and time as covariates and corrected for false discovery rate (FDR). When controlling for these relevant covariates, we observed significant differences between IF-P and CR for 15 metabolites (Fig.  3a , Supplementary Table  S4 ): 2,3-dihydroxybenzoic acid, malonic acid, choline, agmatine, protocatechuic acid, myoinositol, oxaloacetic acid, xylitol, dulcitol, asparagine, n-acetylglutamine, sorbitol, cytidine, acetylcarnitine, and urate ( p .adj ≤ 0.089). To estimate the univariate classification performance of the 15 significant metabolites, we performed a receiver operating characteristic (ROC) analysis. Ten metabolites demonstrated a moderate area under the curve (AUC) (0.718–0.819), while five metabolites had an AUC < 0.70. Therefore, to improve classification performance, we constructed a supervised PLS-DA model using levels of the 15 significant metabolites ( p .adj ≤ 0.089) and analyzed variable importance in projection (VIP) scores (Supplementary Fig.  S3b ). Five metabolites with a VIP > 1.0 (2,3-dihydroxybenzoic acid, malonic acid, protocatechuic acid, agmatine, and myoinositol) were retained to construct an enhanced orthogonal projection to latent structures discriminant analysis (OPLS-DA) model. In contrast, the model fit was assessed with 100-fold leave-one-out cross-validation (LOOCV; see “Methods” section). Permutation testing showed the refined OPLS-DA model to have an acceptable fit to data ( Q 2  = 0.460, p  < 0.001), with appreciable explanatory capacity ( R 2  = 0.506, p  < 0.001; Supplementary Fig.  S3c ). The ROC analysis produced an area under the curve (AUC) of 0.929 (95% CI: 0.868–0.973, sensitivity = 0.8, specificity = 0.9; Supplementary Fig.  S3d ) between the CR and IF-P groups showing good accuracy of the GLM and providing strong support for the differential expression of these 15 metabolites between groups.

a Abundance and log fold-change of significant plasma metabolites between IF-P and CR groups as determined by a general linear model (GLM) adjusted for age, sex, and time. All GLM analyses utilized two-sided p -values, with multiple testing corrections applied using the Benjamini–Hochberg method ( p .adj). Metabolome pathway analysis was conducted for b IF-P and c CR using all reliably detected metabolites showing significantly altered pathways ( p .adj < 0.10) with moderate and above impact (>0.10). Impact scores were calculated using a hypergeometric test, while significance was assessed via a test of relative betweenness centrality, emphasizing the changes in metabolic network connectivity. For all panels, IF-P: n  = 20, CR: n  = 19. Source data are provided as a Source Data file.

Two metabolites, malonic acid, and acetylcarnitine, increased compared to the CR intervention. Several other investigators have noted the increase in acetylcarnitine via fasting protocols 43 , 44 . This increase is consistent with free fatty acid mobilization and increased transportation of these fatty acids via carnitine acylation into the mitochondria for fatty acid oxidation. These results would also be consistent with the expected ketogenesis, although not documented in our study, but noted by similar fasting interventions 44 . Relatedly, malonic acid, a naturally occurring organic acid, is a key regulatory molecule in fatty acid synthesis via its conversion to acetoacetate; hence, our results may reflect this increased synthesis in response to the mobilization and oxidation of fatty acids occurring during fasting. Other metabolites that decreased with IF-P include several sugar alcohols (myoinositol, dulcitol, and xylitol). Dulcitol (galactitol) is a sugar alcohol derived from galactose. It is possible that during fasting, levels of dulcitol decrease as glucose (initially) and free fatty acids (after 24–36 h of fasting) are preferentially utilized as energy substrates. One amino acid (asparagine) and one amino acid analog (N-acetylglutamine, associated with consumption of a Mediterranean diet 45 ) also decreased with IF-P relative to CR. Finally, 2,3-dihydroxybenzoic acid significantly decreased with IF-P. This metabolite is formed during the metabolism of flavonoids, as it is found abundantly in fruits, vegetables, and some spices. At the cellular level, this hydroxybenzoic acid functions as a cell signaling agent and has been speculated as a potentially protective molecule in various cancers 46 . It is unclear whether this metabolite decreased due to either dietary intake or metabolic processes related to high-protein intake or the fasting protocol. Collectively, the metabolic responses to these dietary regimens reflect the interrelationships of several anabolic and catabolic physiologic responses to three key components of these interventions: (a) the WL process itself, (b) changes in amount (and type) of macronutrient distribution (i.e., meal replacement shakes vs. whole food diet approach; higher vs. normal protein intakes), and (c) the adherence to fasting (IF-P only).

To determine the significantly impacted pathways of the dietary interventions, we grouped participant samples according to baseline or intervention period (weeks four and eight), with IF-P and CR assessed separately. A total of 14 pathways were significant in the IF-P group ( p .adj < 0.10; Fig.  3b ), with three displaying large impact coefficients (>0.5): (1) Glycine, serine, and threonine metabolism, (2) alanine, aspartate, and glutamate metabolism, and (3) ascorbate and aldarate metabolism. In comparison, 24 pathways were significant for the CR group (Fig.  3c ), with four showing large impact coefficients (>0.5): (1) Phenylalanine, tyrosine, and tryptophan biosynthesis, (2) alanine, aspartate, and glutamate metabolism, (3) citrate cycle (TCA cycle), and (4) glycine, serine and threonine metabolism. Notably, the glycine, serine, and threonine pathway has recently been found in preclinical models to play a pivotal role in longevity and related life-sustaining mechanisms independent of diet, though heavily impacted by fasting time and caloric restriction 47 . This may be partially related to the ability of glycine to increase tissue glutathione 48 , 49 and protect against oxidative stress 50 . In our analysis, this pathway was significant in both diet groups and is biochemically and topologically related to the additionally captured amino acid pathway, alanine, aspartate, and glutamate metabolism, as well as the energy-releasing pathway, the citrate cycle (TCA cycle). Notably, in the CR group, phenylalanine, tyrosine, and tryptophan biosynthesis, are important for neurotransmitter production and reported to be suppressed (tryptophan) in obesity 51 . This representation may have also been attributed to the differences in protein intake 52 or differences in dietary diversity 53 , yet to be determined. Regardless, we noted similar representations of pathway impact between IF-P and CR, with metabolic response centered on utilization of amino acids in addition to lipid turnover and energy pathways.

Gut microbiome and plasma metabolome latent factors indicate differential multi-omic signatures between IF-P and CR regimens

As the plasma metabolome has been suggested as a bidirectional mediator of GM influence on the host 54 , we performed a multi-omics factor analysis (MOFA) 55 to identify potential patterns of covariation and co-occurrence between the microbiome and circulating metabolites. Operating in a probabilistic Bayesian framework, MOFA simultaneously performs unsupervised matrix factorization to obtain overall sources of variability via a limited number of inferred factors and identifies shared versus exclusive variation across multiple omic data sets 55 . Eight latent factors were identified (minimum explained variance ≥2%; see “Methods” section), with the plasma metabolome and GM explaining 37.12% and 17.49% of the overall sample variability, respectively (Fig.  4a ). Based on significance and the proportion of total variance explained by individual factors for each omic assay, Factors 1 ( R 2  = 11.98) and 6 ( R 2  = 5.28) captured the greatest covariation between the two omic layers (Fig.  4a ; Supplementary Table  S5 ). In contrast, Factors 2 and 5 were nearly exclusive to the metabolome, and factors 3 and 4 to the GM. Interestingly, Factor 1 was significantly negatively correlated to dietary protein intake (Spearman rho = −0.270, p.adj = 0.021; Fig.  4b ) and captured the variation associated with the CR diet (Wilcoxon rank-sum test, p .adj = 3.2e-04; Fig.  4c ). Factor 6 had the greatest number of significant correlations, including negative associations with visceral adipose tissue, waist circumference, body weight, BMI, fat mass, android fat, subcutaneous adipose tissue, dietary sodium, carbohydrate, fat, energy intake (kcal), and sugar (Spearman rho ≤ −0.220, p .adj ≤ 0.075) and captured the variation associated with IF-P (Wilcoxon rank-sum test, p .adj = 0.007).

a The cumulative proportion of total variance explained ( R 2 ) and proportion of total variance explained by eight individual latent factors for each omic layer. b Spearman correlation matrix of the eight latent factors and clinical anthropometric and dietary covariates. Each circle represents a separate association, with the size indicating the significance (-log10 ( p -values)) and the color representing the effect size (hue) with its direction (red: positive; blue: negative). All correlations are calculated using two-sided tests. Asterisks within a circle denote significance after adjustment with the Benjamini–Hochberg method. c Scatter plot of Factors 1 and 6, with each dot representing a sample colored by intervention. Box and whisker plots illustrate significant differences between groups after adjusting for multiple testing using the Benjamini–Hochberg method (Wilcoxon rank-sum test; top = Factor 1, p .adj = 3.2e-04; right = Factor 6, p .adj = 0.007). The plots show boxes ranging from the first to the third quartile and the median at the center, with whiskers extending to the minimum and maximum values. d Factor 1 and 6 loadings of genera and metabolites with the largest weights annotated. Symbols: * p .adj < 0.10, ** p .adj < 0.01, *** p .adj < 0.001, **** p .adj < 1.0e-04. For all panels, IF-P: n  = 20, CR: n  = 19. Source data are provided as a Source Data file.

Assessing the positive weights (feature importance) of Factor 1 revealed a microbial and metabolomic signature linked with CR, including the taxa Faecalibacterium , Romboutsia , and Roseburia , and the plasma metabolites myoinositol, agmatine, N-acetylglutamine, erythrose, and mucic acid (Fig.  4d ). Previous dietary restriction studies have reported co-occurrence of gut microbial taxa and plasma metabolites that span a wide variety of applications and investigations 56 . The specific co-occurrences observed in Factor 1 exhibited an abundance of butyrate-producing bacterial taxa that utilize carbohydrates as their predominant substrate and plasma metabolites that are generally involved in carbohydrate metabolism, such as erythrose, an intermediate in the pentose phosphate pathway (PPP), and mucic acid which is derived from galactose and/or galactose-containing compounds (i.e., lactose). These co-occurrence patterns biologically cohere considering the nutritional profile of the CR group and the large contribution of fiber-rich, unrefined carbohydrates and reduction in sugar (~50% kcal from sugar). Indeed, these nutritional changes may have influenced the GM to accommodate changes in dietary substrate more efficiently. One interesting co-occurrence was the genus Romboutsia and metabolite N-acetylglutamine. Romboutsia has been shown to produce several SCFAs and ferment certain amino acids, including glutamate 57 . N-acetylglutamine is biosynthesized from glutamate; thus, its co-occurrence with the abundance of Romboutsia encourages further exploration into this interaction 58 .

Factor 6 captured the signature associated with IF-P, with positive contributions from the taxa Incertae Sedis ( Ruminococcaceae family), Erysipelatoclostridium , Christensenellaceae R-7 group , Oscillospiraceae UCG-002, and Alistipes , and the plasma metabolites malonic acid, adipic acid, succinate, methylmalonic acid, and mucic acid (Fig.  4d ). Prior work has established that Alistipes increases from diets rich in protein and fat, and contributes to the highest number of putrefaction pathways (i.e., fermentation of undigested proteins in the GI tract) over the other commensals 59 . This could explain the co-occurrence of plasma metabolites from protein catabolism, such as 2-aminoadipid acid, adipic acid, and glutamic acid 22 , 59 . Oscillospiraceae has recently been viewed with next-generation probiotic potential, harboring positive regulatory effects in areas related to obesity and chronic inflammation 60 . Mentioned prior, recent studies have reported on the role of Christensenellaceae on human health, participating in host amino acid and lipid metabolism as well as fiber fermentation 20 , with Christensenellaceae R-7 group notably evidenced to correlate with visceral adipose tissue reduction 22 . As such, the elevated abundance of microbes in the GM of IF-P participants observed in this study in tandem with the co-occurrence of metabolites indicative of protein degradation and mobilization and oxidation of fatty acids, such as methylmalonic acid, malonic acid, and succinate, presents a nascent multi-omic signature of IF-P. In addition, and more pronounced in the IF-P vs CR group, participants decreased sugar intake by ~75% (kcals) compared to baseline levels. Considering the other regimental components of IF-P, the differences in multi-omic signatures likely display the selective pressures of these two interventions.

Gut microbiome (GM) composition is associated with weight loss (WL) responsiveness to IF-P diet

The IF-P intervention produced a microbiome and metabolomic response; however, the loss in body weight and fat across individuals varied (Fig.  5a ). To provide deeper characterization and explore differential features of WL responsiveness, we performed a GM-focused subgroup analysis by employing shotgun metagenomic and untargeted fecal metabolomic surveys in 10 individuals that either achieved ≥10% loss in body weight or bordered on clinically important WL (i.e., >5% BW; herein, ‘High’ and ‘Low’ responders) 61 . Importantly, baseline characteristics between WL responder classification did not differ significantly (baseline body weight: High, 108.9 ± 30.8 vs. Low, 81.9 ± 18.1 kg, p  = 0.117; Supplementary Table  S6 ). Assessing the GM at the fundamental taxonomic rank, species composition showed significant separation by weight loss response evaluated by Bray-Curtis dissimilarity (group × time: R 2  = 0.114, p  = 0.001; Fig.  5b ; Supplementary Table  S7 ), with most of the variation explained by the individual ( R 2  = 0.711, p  = 0.001). In comparison, species level alpha diversity did not differ significantly between classifications (group × time: p  ≥ 0.674; Fig.  5c, d ). Identifying 212 species after filtering, we noted significant differences in bacterial abundances between groups over time (Fig.  5e ; Supplementary Data  8 ). A total of 10 features increased in the High-responder group relative to the Low-response group over the eight-week study period, including Collinsella SGB14861 , Clostridium leptum , Blautia hydrogenotrophica , and less typified species; GGB74510 SGB47635 (unclassified Firmicutes), GGB3511 SGB4688 (unclassified Firmicutes), Faecalicatena contorta , Lachnospiraceae bacterium NSJ-29 , Phascolarctobacterium SGB4573 , GGB38744 SGB14842 (unclassified Oscillospiraceae ), and Massiliimalia timonensis (effect size ≥ 1.163, p .adj ≤ 0.092). The increase in Collinsella , a less characterized anaerobic pathobiont that produces lactate and has been associated with low-fiber intakes 62 , 63 and lipid metabolism 64 , may have been related to the periods of CR and IF, in conjunction with the greater influx of host-released fatty acids in the High-responder group. Relatedly, Clostridium leptum growth has been linked with increases in monounsaturated fat intake, reductions in blood cholesterol 65 , and stimulation of Treg induction (i.e., anti-inflammatory) 66 . The latter association is relevant to the SCFA-promoting (primarily butyrate) qualities of Clostridium leptum 67 . Blautia hydrogenotrophica , an acetogen with bidirectional metabolic cross-feeding properties (e.g., transfer of hydrogen and acetate), is also important for butyrate formation 68 . Taxa that decreased relative to the Low-responder group; Eubacterium ventriosum , Streptococcus salivarius , Eubacterium rectale , Anaerostipes hadrus , Roseburia inulinivorans , Mediterraneibacter glycyrrhizinilyticus , and Blautia massiliensis (effect size ≤ −1.690, p .adj ≤ 0.078), included butyrate producers, Eubacterium ventriosum , Eubacterium rectale , Roseburia inulinivorans , and others, such as Streptococcus salivarius , a nuclear factor kappa B (NF-κB) activity repressor 69 and Peroxisome proliferator-activated receptor gamma (PPARγ) inhibitor potentially influencing lipid and glucose metabolism 70 . Investigating monozygotic (MZ) twin pairs, Eubacterium ventriosum was more abundant in the higher BMI siblings 26 , with enhanced scavenging fermentation capabilities 71 . Roseburia inulinivorans is a mobile firmicute (flagella) that harbors a wide-ranging enzymatic repertoire able to act on various dietary polysaccharide substrates suggestive of the ability to respond to the availability of alternative dietary substrates 72 . While we noted a more variable shift in fecal total SCFAs, acetate, propionate, butyrate, or valerate (via targeted GC–MS), in the Low weight loss responders, there was no significant difference when compared to High weight loss responders (Wilcoxon rank-sum test, p  ≥ 0.210; Supplementaryl Fig.  S4a ; Supplementary Data  9 ).

a Relative weight loss over the eight-week intervention for each participant in the IF-P group. b NMDS ordination showed the personalized trajectories of participants’ microbiomes over time. Dotted lines connect the same individual and point toward the final sample collection. No significant time or group × time interaction effects for alpha diversity metrics, c observed species, and d the Shannon index. Box and whiskers plots display the box ranging from the first to the third quartile, and the center the median value, while the whiskers extend from each quartile to the minimum or maximum values. Volcano plots displaying differential abundance between High and Low weight loss responders for e microbial species and f functional pathways. Significant features were more enriched in High and Low weight loss responders colored orange and light blue, respectively. g Alluvial plot displaying the fecal metabolite profile at the subclass level (Human Microbiome Database). Most abundant metabolite subclasses displayed (i.e., ≥1%). Metabolome pathway analysis for h High and i Low weight loss responders using all reliably detected fecal metabolites showing altered pathways with moderate and above impact (>0.10). Impact was calculated using a hypergeometric test, while significance was determined using a test of relative betweenness centrality. j Grid-fused least absolute shrinkage and selection operator (GFLASSO) regression of species from differential abundance analysis displayed correlative relationships with fecal metabolites. Species with greater abundance in High (High > Low) and Low (Low > High) weight loss responders are separate‘. For all panels, High: n  = 5, Low: n  = 5. Source data are provided as a Source Data file.

Less affected compared to taxonomic features were the 275 microbial-affiliated metabolic pathways identified after filtering, of which gluconeogenesis III and guanosine ribonucleotides de novo biosynthesis were increased (effect size ≥ 0.108, p .adj = 0.079), while super pathway of L-alanine biosynthesis, sucrose degradation IV (sucrose phosphorylase), sucrose degradation III (sucrose invertase), super pathway of thiamine diphosphate biosynthesis III, and flavin biosynthesis I (bacteria and plants) were decreased in the High relative to the Low weight loss responder group (effect size ≤ −0.247, p .adj ≤ 0.079; Fig.  5f ; Supplementary Data  10 )

As the difference in microbial shifts versus function is well established, we also tracked the fecal metabolome to better understand metabolic modification/production and identify potential microbial metabolic targets for future weight loss interventions. Overall, we reliably detected (QC relative standard deviation > 20% and mean intensity value > 1000 in 80% of samples) and annotated 607 (Human Metabolome Database) compounds across fecal samples. Notably, we found the fecal metabolite profile of both subgroups abundant in amino acids, peptides, and analogs, with decreases in sulfates, furanones, and quaternary ammonium salts and increases in cholestane steroids, carboxylic acid derivatives, and imidazoles (Fig.  5g ). Assessing metabolite changes between groups did not yield significance when comparing logFC values (Wilcoxon rank-sum test, p .adj > 0.10; Supplementary Fig.  S4b ). Pathway analysis of High weight loss responders revealed prominent metabolic signatures relevant to lipid metabolism (glycerolipid and arachidonic metabolism), nucleotide turnover (pyrimidine metabolism), and aromatic amino acid formation (phenylalanine, tyrosine, and tryptophan biosynthesis; Fig.  5h , Supplementary Data  11 ). In comparison, the more prominent enriched pathways for Low weight loss responders included those related to amino acid and peptide metabolism (glycine, serine, and threonine, d-glutamine and d-glutamate, and tyrosine metabolism and arginine biosynthesis; Fig.  5i , Supplementary Data  12 ).

Finally, species captured by our differential abundance analysis were channeled into a GFLASSO model with the fecal metabolome library to select metabolically relevant compounds best predicted by microbial abundances. Restricting taxa and metabolites displaying stronger co-occurrence signals (GFLASSO coefficients > 0.02), we noted several patterns (Fig.  5j ). This included positive associations between GGB3511 SGB4688 (unclassified Firmicute) and malonic acid (important to fatty acid metabolism), as well as Roseburia inulinivorans and 3-Hydroxy-2-oxo-1H-indole-3-acetic acid. Negative associations included Phascolarctobacterium SGB4573 with the fatty acid ester, methyl sorbate, and Streptococcus salivarius (anti-inflammatory) with leukotriene B4 dimethylamide.

Differences detected in our subgroup analysis suggest that the GM composition plays a role in WL responsiveness during IF-P interventions. Notable differences in taxa and fecal metabolites suggest differing substrate utilization capabilities and nutrient-acquiring pathways between High and Low responders, despite being on the same dietary regimen. Although differences between High and Low responders were statistically significant for the microbiome data, the magnitude of differences varied, suggesting further research is needed to clarify these differences.

Long-term IF-P remodels the gut microbiome after substantial weight loss – A case study

Considering the microbiomic and metabolic importance of sustained WL, we additionally performed a longitudinal, exploratory case study analysis on the participant who lost the most body weight during the eight-week WL period (−15.3% BW, −24.9 kg). Under rigorous clinical supervision, this individual was guided through and comprehensively tracked over 52 weeks, strictly adhering to an IF-P regimen, including WL (0–16 weeks) and maintenance (16–52 weeks) periods, which included adjusting the calorie intake to maintain energy balance. Microbial richness and evenness at the species level displayed a general inverse trend with body weight reduction, although they converged at 52 weeks (Fig.  6a, b ). Species dissimilarity peaked at weeks four and 16, after which it plateaued, but remained consistently higher in comparison to baseline over the 52-week period (Fig.  6c ). Examining positive linear coefficients of a PERMANOVA model, constructed to detect variation between community compositions over time, dominant influences included several species within the Lachnospiraceae family such as Fusicatenibacter saccharivorans , Blautia wexlerae , Blautia massillensis , Anaerostipes hadrus , and Coprococcus comes and others like Akkermansia muciniphila (Fig.  6d ). Negative contributions included species from the Oscillospiraceae family, such as Ruminococcus bromii and Ruminococcus torques . Indeed, visualizing community composition over the sampling time points suggested specific GM remodeling (Fig.  6e ; Supplementary Data  13 ). Many keystone taxa prominent over time in the microbiome are highly relevant to the significant reduction in body weight and metabolic improvement of the case-study participant. For example, Blautia wexlerae , a commensal bacterium recently reported to confer anti-adipogenesis and anti-inflammatory properties to adipocytes 73 became visually more prominent over time. This association was also the case for the health-associated microbe, Anaerostipes hadrus , which converts inositol stereoisomers (including myoinositol) to propionate and acetate, apt to improve insulin sensitivity and reduce serum triglyceride levels 74 , translating to reduced host metabolic disease risk 75 . Other elevated taxa, like the mucin-degrading Akkermansia muciniphila and Bacteroides faecis , are negatively correlated with markers for insulin resistance 76 . There was also a notable bloom of Collinsella SGB14861 (anaerobic pathobiont producing lactate) 63 and suppression of Eubacterium rectale , Ruminococcus torques (associated with circadian rhythm disruption in mice) 77 , and Ruminococcus bromii (an exceptional starch degrader) 78 .

Change in alpha diversity metrics a observed species and b Shannon index with percentage of baseline body weight. c Bray-Curtis dissimilarity at the species level with d top PERMANOVA model coefficients (analysis: species~time). e Alluvial plot displaying the variation in abundance of the 20 most prevalent bacteria over time. For visual clarity, the less abundant taxa are not displayed. f Canberra distance of fecal metabolome with g top PERMANOVA model coefficients (analysis: pathway~time). h Pathway analysis of fecal metabolites comparing baseline to subsequent sample collections. Data are plotted as -log10(p) versus pathway impact. Node size corresponds to the proportion of metabolites captured in each pathway set, while node color signifies significance. Impact was calculated using a hypergeometric test, while significance was determined using a test of relative betweenness centrality. No p -value adjustments were made. Source data are provided as a Source Data file.

Compared to the more pronounced shifts in the GM, an inspection of Bray-Curtis dissimilarity at the microbial metabolic pathway level was much less affected (Supplementary Fig.  S5a ). Though positive contributions in multiple biosynthesis pathways were noted, as well as reductions in the superpathway of UDP-glucose-derived O-antigen building blocks biosynthesis and glucose and glucose-1-phosphate degradation (Supplementary Fig.  S5b ; Supplementary Data  14 ). We also tracked the fecal metabolome concordance with the GM to corroborate potential metabolic output. Shifts in metabolites captured by calculating the Canberra distance were prominent (Fig.  6f ), with positive influences from agrocybin (possessing antifungal activity 79 ), nicotinic acid (nicotinamide adenine dinucleotide precursor), and sulfate, and reductions in cadaverine (involved in the inhibition of intestinal motility 80 ), maltitol, acetohydroxamic acid (a urease inhibitor), and hypoxanthine, after removing the dominant amino acid subclass (Fig.  6g ; Supplementary Fig.  S5c ). At the chemical class level, we observed apparent shifts in chemical subclasses; cholestane steroids, amines, purines, and purine derivatives, and amino acids, peptides, and analogs (Supplementary Fig.  S5d ). Given our case-study approach, we performed a pathway analysis using all reliably detected fecal metabolites at each collection point over 52 weeks. Pathway analysis (Fig.  6h ) identified primary bile acid biosynthesis ( p  = 0.014) and cysteine and methionine metabolism ( p  = 0.096) as having the greatest significance, while the greatest impact (I) was observed in phenylalanine, tyrosine, and tryptophan biosynthesis and linoleic acid metabolism ( I  = 1.0). Alanine, aspartate, and glutamate metabolism ( I  = 0.756), vitamin B6 metabolism ( I  = 0.647), sulfur metabolism ( I  = 0.532), phenylalanine metabolism (I =  0.357), and nicotinate and nicotinamide metabolism ( I  = 0.194) also displayed marked pathway impacts (Supplementary Fig.  S5e ; Supplementary Data  15 ). Together, these integrated findings from the group comparisons (IF-P vs. CR), high vs. low responders, and the case study, suggest that the remodeling of the gut microbiome through sustained weight loss on an IF-P regimen not only alters the microbial composition but also influences key metabolic pathways and output, reflective of fat mobilization and metabolic improvement.

Our study demonstrates distinct effects of IF-P on gut symptomatology and microbiome, as well as circulating metabolites compared to continuous CR. We observed significant changes in the GM response to both interventions; however, the IF-P group exhibited a more pronounced community shift and greater divergence from baseline (i.e., intra-individual Bray-Curtis dissimilarities). This shift was characterized by increased specific microbial families and genera, such as Christensenellaceae , Rikenellaceae , and Marvinbryantia , associated with favorable metabolic profiles. Furthermore, IF-P significantly increased circulating cytokine concentrations of IL-4, IL-6, IL-8, and IL-13. These cytokines have been linked to lipolysis, WL, inflammation, and immune response. The plasma metabolome analysis revealed distinct metabolite signatures in IF-P and CR groups, with the convergence of multiple metabolic pathways. These findings shed light on the differential effects of IF regimens, including IF-P as a promising dietary intervention for obesity management and microbiotic and metabolic health.

While acknowledging individual contributions of WL, protein pacing, and IF, we propose that the beneficial shifts observed may be best characterized as the culmination of features inherent in our IF-P approach. For example, it is possible that microbial competition is leveraged during reduced and intermittent nutritional input periods, emphasizing nutrient composition and food matrix type (combination of whole food and meal replacements vs. primarily whole food), affecting available substrates for gut microbes. IF-P participants’ fiber intake was concentrated in fiber-rich (RS5 type) shakes, offering immediate availability of fiber to the GI tract. In contrast, CR participants consumed fiber through whole foods, leading to a slower digestion and absorption process influenced by individual digestive transit times and enzymatic profiles. This nutritional environment may create ecological niches that support symbiont microbial communities. In this investigation, we provide support of such remodeling, with intentional fasting and increased relative protein (protein pacing) consumption well-validated to improve body composition and metabolism during weight loss 7 , 8 , 15 . Our results align with previous studies on CR, where greater relative protein intake was associated with an increased abundance of Christensenella 81 . This increase is likely a result of increased amino acid-derived metabolites 21 . We also observed increased signatures of amino acid metabolism in the GM of IF-P participants, which may be attributed to increased nitrogen availability, prompting de novo amino acid biosynthesis. The liquid format of two of the daily meals and precise timing of high-quality protein consumption (Protein Pacing) in the IF-P regimen may have influenced these results, as amino acids play essential roles in microbial communities, acting as energy and nitrogen sources and essential nutrients for amino acid auxotrophs.

In addition to the differences in nutrient composition, the IF-P group exhibited a profound reduction (33%) in visceral fat 15 . This reduction is significant because visceral fat is highly correlated with GM. While the specific influence of GM on fat depots in our study remains unclear, the shift in cytokine profile and metabolic pathways suggests an interaction between GM and fat metabolism. Regarding GM-host interaction, we did not detect changes in gut permeability assaying LBP. However, correlations were found with cytokines IL-4 and IL-13 and microbes Colidextribacter (negative association) and Ruminoccus gauveauii group (positive association). These associations may reflect the direct impact of the dietary intervention, yet they also hint at a deeper crosstalk within the gut-immune axis. This crosstalk is known to play a pivotal role in modulating host inflammation and influencing adipose tissue signaling pathways 42 . Furthermore, the observed microbial shifts, including changes in populations of Christensenella , suggest a nuanced role for certain microbes in regulating metabolic health. Notably, certain strains of Christensenella have been implicated in the regulation of key metabolic markers, such as glycemia and leptin levels, and in promoting hepatic fat oxidation 82 .

Our findings also underscore that GM composition plays a role in WL responsiveness during IF-P interventions. Subgroup analysis based on WL responsiveness revealed significant differences in species composition at the taxonomic level. The High-responder group showed an increased abundance of certain bacteria associated with metabolic benefits and anti-inflammatory effects. In contrast, the Low-responder group exhibited an increased abundance of butyrate-producing and nutritionally adaptive species (e.g., Eubacterium ventriosum 71 and Roseburia inulinivorans 72 ). Fecal metabolome analysis further highlighted differences between the two subgroups, with distinct metabolic signatures and enrichment in specific metabolic pathways. Notably, the High WL responders displayed enrichment of fecal metabolites involved in lipid metabolism. In contrast, Low responders were more prominent in pathways related to the metabolism of amino acids and peptides, including glycine, serine, and threonine, d-glutamine, and d-glutamate, as well as tyrosine metabolism and arginine biosynthesis. The latter metabolic signature has been reported in individuals with severe obesity undergoing high-protein, low-calorie diets 83 . As both High and Low WL responders were consuming the same diet, our results suggest differences in GM composition and metabolism, which could play a role in determining the success of an IF-P regimen. Though, as these enrichment analyses were performed in an exploratory manner, we acknowledge the need for a more systematic approach to validate these findings.

Finally, we provide evidence of long-term GM stabilization from these changes by following one individual over 12 months. Dietary restriction is widely used to reduce fat mass and weight in individuals with or without obesity; however, weight regain after such periods presents a critical challenge, and the underlying homeostatic mechanisms remain largely elusive. Notably, keystone taxa that became more prominent over time were associated with anti-adipogenesis, improved insulin sensitivity, and reduced metabolic disease risk. The microbial shifts were accompanied by noticeable changes in the fecal metabolome, with shifts in various metabolites and chemical subclasses. Pathway analysis identified impacts on primary bile acid biosynthesis, cysteine and methionine metabolism, and other fat mobilization and metabolic improvement pathways. These shifts were accompanied by noticeable changes in the fecal metabolome, particularly in metabolites and chemical subclasses related to lipid metabolism, nucleotide turnover, and aromatic amino acid formation.

Despite the valuable insights from our study on the complex interactions between intermittent fasting, higher protein intake using protein pacing, the GM, and circulating metabolites in obese individuals, several limitations should be acknowledged. First, our reliance on fecal samples to represent the GM may have overlooked potential microbial populations in the upper GI tract. Including samples from proximal regions in future studies would provide a more comprehensive understanding of the gut microbiome’s response to IF-P and CR. In addition, the sample size for our study was determined based on the primary outcomes related to body weight and composition from the parent study 15 . This sample size may have reduced statistical power and potentially amplified individual variability among participants. However, it is important to note that the smaller RCT design allowed for more precise control over diet and lifestyle factors, minimizing potential confounding influences on the study outcomes. Furthermore, the study’s duration was limited to eight weeks, which prevented potential insights into the differential long-term effects between the two interventions. However, we were able to extend the follow-up duration and conduct periodic assessments for a year in our case-study participant, offering a more comprehensive understanding of the sustainability of the observed changes and the potential for weight regain for IF-P. The current study compared a combination of whole food and supplements (shakes and bars; IF-P) versus primarily whole food (CR), which together with variations in protein and fiber content and type may have influenced the gut symptomatology and nutrient absorption between groups. Additionally, study participants self-reported dietary intake daily, although there was close monitoring of intake through the return of empty food packaging/containers of consumed food and daily monitoring by investigators and weekly meetings with a registered dietitian. Overall, knowledge gaps are present in this research, including how the microbiome is rebuilt after food reintroduction and how overall caloric restriction and specific macronutrients contribute to this process. However, considering the multifactorial nature of weight loss and metabolic health, our work represents an important precedent for future work. Future investigators should consider integrating these factors to provide a more comprehensive understanding of the underlying mechanisms. Additional research is warranted to characterize the metabolic signature of IF-P, the time relationship between these fasting periods, and the analysis of these metabolic changes. A strength of our High-Low-responder and case-study analyses is the hypothesis-driving nature of the findings, from which targeted microbiome and/or precision nutrition interventions can be designed and tested.

In conclusion, our study provides valuable insights into the complex interactions among intermittent fasting and protein pacing, the GM, and circulating metabolites in individuals with obesity. Specifically, intermittent fasting - protein pacing significantly reduces gut symptomatology and increases gut microbes associated with a lean phenotype ( Christensenella ) and circulating cytokines mediating total body weight and fat loss. These findings highlight the importance of personalized approaches in tailoring dietary interventions for optimal weight management and metabolic health outcomes. Further research is necessary to elucidate the underlying mechanisms driving these associations and to explore the therapeutic implications for developing personalized strategies in obesity management. Additionally, future studies should consider investigating microbial populations in upper GI sections and potential intestinal tissue remodeling to gain a more comprehensive understanding of the gut microbiome’s role in these interventions.

Study design and participants

The protocol of the clinical trial was registered on March 6, 2020 (Clinicaltrials.gov; NCT04327141), and the results of the primary analysis have been published previously 15 . Briefly, participants were recruited from Saratoga Springs, NY, and were provided informed written consent in accordance with the Skidmore College Human Subjects Institutional Review Board before participation (IRB#: 1911-859), including consent for the use of samples and data from the current study. Each procedure performed was in adherence with New York state regulations and the Federal Wide Assurance, which follows the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research, and in agreement with the Helsinki Declaration (revised in 1983). Their physicians performed a comprehensive medical examination/history assessment to rule out any current cardiovascular or metabolic disease. For at least six months before the start of the study, all eligible participants were either sedentary or lightly active (<30 min, two days/week of organized physical activity), with overweight or obesity (BMI > 27.5 kg/m2; % body fat > 30%), weight stable (±2 kg), and middle-aged (30–65 years). In addition, participants taking antibiotics, antifungals, or probiotics within the previous two months were excluded. Enrolled participants were matched for body weight, BMI, and body fat and randomly assigned to one of two groups: (a) IF-P ( n  = 21; 14 women; 7 men) or (b) CR ( n  = 20; 12 women; 8 men) for eight weeks. During a one-week run-in period, subjects maintained a stable body weight by consuming a similar caloric intake as their pre-enrollment caloric intake while maintaining their sedentary lifestyle. This was confirmed by matching their pre-enrollment dietary intake to the one-week run-in diet period 15 . Following baseline testing, participants were provided detailed instructions on their weight loss dietary regimen (Supplementary Table  S1 ) and received weekly dietary counseling and compliance/adherence monitoring from the research team via daily food records, and weekly registered dietitian meetings, along with weekly visits to the Human Nutrition and Metabolism laboratory at Skidmore College (Saratoga Springs, NY) for meal distribution and empty packet/container returns. All outcome variables were assessed pre (week 0), mid (week 4), and post (week 8). All participants were compensated $100 for successful completion of the study and received an additional monthly stipend of $75 for groceries (CR group only) or up to two meals per day of food supplements and meal replacements (IF-P only).

IF days consisted of ~350–550 kcals per day, in which participants were provided a variety of supplements and snacks. Protein pacing (P) days for IF-P consisted of four and five meals/day for women and men, respectively, two of which (breakfast and one other meal) were liquid meal replacement shakes with added whole foods (Whole Blend IsaLean® Shakes, 350/400 kcals, 30/36 g of protein/meal, 9 g of fiber); a whole food evening dinner meal (450/500 kcals men), an afternoon snack (200 kcals, men only), and an evening protein snack (IsaLean® or IsaPro® Shake or IsaLean Whole Blend® Bar; 200–250 kcals). This dietary regimen provided 1350–1500 and 1700–1850 kcals/day for women and men, respectively, and a macronutrient distribution targeting 35% protein, 35% carbohydrate, 20–30 g/day of fiber, and 30% fat. Isagenix International, LLC (Gilbert, AZ, USA) provided all meal replacement shakes, bars, beverages, and supplements. In comparison, participants assigned to the CR diet followed specific guidelines of the National Cholesterol Education Program Therapeutics Lifestyle Changes (TLC) diet of the American Heart Association with a strong Mediterranean diet influence of a variety of fresh vegetables, fruits, nuts, and legumes. The specific macronutrient distribution recommended was <35% of kcal as fat; 50%–60% of kcal as carbohydrates; 15% kcal as protein; <200 mg/dL of dietary cholesterol; and 20–30 g/day of fiber. The total calorie intake was 1200 and 1500 calories per day for women and men, respectively, during the 8-week weight loss intervention. In addition to weekly meetings with the registered dietitian and daily contact with research team members, subjects were provided detailed written instructions for their meal plans. They were closely monitored through daily participant-researcher communication (e.g., email, text, and mobile phone), two-day food diary analysis, weekly dietary intake journal inspections, weekly meal/supplement container distribution, and returning empty packets and containers.

Gastrointestinal (GI) symptom rating scale

Participants completed the 15-question GI symptom rating scale (GSRS) 84 at baseline, week four, and week eight. Briefly, each question is rated on a 7-point Likert scale (1 = absent; 2 = minor; 3 = mild; 4 = moderate; 5 = moderately severe; 6 = severe and 7 = very severe) and recalled from the previous week. Questions include symptoms related to upper abdominal pain, heartburn, regurgitation (acid reflux), empty feeling in the stomach, nausea, abdominal rumbling, bloating, belching, flatulence, and questions on defecation. The GSRS questionnaire provides explanations of each symptom, is understandable, and has reproducibility for measuring the presence of GI symptoms 85 . In our analysis, a score of ≥2 (minor) was defined as symptom presence, and a score ≥ 4 (moderate) was defined as moderate symptom presence. Furthermore, to better categorize symptom location, bloating, flatulence, constipation, diarrhea, stool consistency, defecation urgency, and sensation of not completely emptying bowels were classified as lower GI symptoms, and nausea, heartburn, regurgitation, upper abdominal pain, empty feeling in the stomach, stomach rumbling, and belching was classified as upper GI symptoms. Total scores were also generated for overall symptom and moderate symptom presence.

Fecal sample collection and DNA extraction

Participants were instructed to provide stool samples at baseline, week four, and week eight of the intervention. The case-study participant additionally provided samples at weeks 12, 16, 32, and 52. The entire bowel movement was collected and transported within 24 h of defecation to the Skidmore College Human Nutrition and Metabolism (Saratoga Springs, NY) laboratory using a cooler and ice packs and frozen at −80 °C. Samples were then sent to ASU (Phoenix, AZ) overnight on dry ice for analysis, where they were thawed at 4 °C and processed. Wet weight was recorded to the nearest 0.01 g after subtracting the weight of fecal collection materials. Stool samples were then rated according to the BSS 86 , homogenized in a stomacher bag, and the pH was measured (Symphony SB70P, VWR International, LLC., Radnor, PA, USA). Next, the extraction of DNA was performed using the DNeasy PowerSoil Pro Kit (Cat. No. 47016, Qiagen, Germantown, MD) per the manufacturer’s instructions. DNA concentration and quality were quantified using the NanoDrop™ OneC Microvolume UV-Vis Spectrophotometer (Thermo Scientific™, Waltham, MA) according to manufacturer instructions. The OD 260 /OD 280 ratio of all samples was ≥1.80 (demonstrating DNA purity).

Quantification of bacterial 16S rRNA genes

To estimate total bacterial biomass per sample (16S rRNA gene copies per gram of wet stool), DNA extracted from the fecal collections was assessed via quantitative polymerase chain reaction (qPCR) based on previously published methods 87 , 88 . Briefly, all 20 μL qPCR reactions contained 10 uL of 2X SYBR Premix Ex Taq ™ (Tli RNase H Plus) (Takara Bio USA, Inc., San Jose, CA, USA), 0.3 μM (0.6 μL) of each primer (926 F: AAACTCAAAKGAATTGACGG; 1062 R: CTCACRRCACGAGCTGAC), 2 μL DNA template (or PCR-grade water as negative control), and 6.8 μL nuclease-free water (Thermo Fisher Scientific, Waltham, MA, USA). PCR thermal cycling conditions were as follows: 95 °C for 5 min, followed by 35 cycles of 95 °C for 15 s, 61.5 °C for 15 s, and 72 °C for 20 s, then hold at 72 °C for 5 min, along with a melt curve of 95 °C for 15 s, 60 °C for 1 min, then 95 °C for 1 s. Quantification was performed using a QuantStudio3™ Real-Time PCR System by Applied Biosystems with QuantStudio Design and Analysis Software 1.2 from Thermo Fisher Scientific (Waltham, MA, USA). All samples were analyzed in technical replicates. For quality assurance and quality control, molecular negative template controls (NTC) consisting of PCR-grade water (Invitrogen, Waltham, MA, USA) and positive controls created by linearized plasmids were run on every qPCR plate. Standard curves were run-in triplicate and used for sample quantification, ranging from 10 7 to 10 1 copies/μL with a cycle threshold (CT) detection limit cutoff of 33. Reaction efficiency was approximately 101%, with a slope of −3.29 and R 2  ≥ 0.99.

Fecal microbiome analysis

Amplification of the 16S rRNA gene sequence was completed in triplicate PCRs using 96-well plates. Barcoded universal forward 515 F primers and 806 R reverse primers containing Illumina adapter sequences, which target the highly conserved V4 region, were used to amplify microbial DNA 89 , 90 . PCR, amplicon cleaning, and quantification were performed as previously outlined 90 . Equimolar ratios of amplicons from individual samples were pooled together before sequencing on the Illumina platform (Illumina MiSeq instrument, Illumina, Inc., San Diego, CA). Raw Illumina microbial data were cleaned by removing short and long sequences, sequences with primer mismatches, uncorrectable barcodes, and ambiguous bases using the Quantitative Insights into Microbial Ecology 2 (QIIME2) software, version 2021.8 91 .

16S rRNA sequencing produced 7,366,128 reads with a median of 53,776 per sample (range: 9512–470,848). Paired-end, demultiplexed data were imported and analyzed using QIIME2 software. Upon examination of sequence quality plots, base pairs were trimmed at position 20 and truncated at position 240 and were run through DADA2 to remove low-quality regions and construct a feature table using ASVs. Next, the ASV feature table was passed through the feature-classifier plugin 92 , which was implemented using a naive Bayes machine-learning classifier, pre-trained to discern taxonomy mapped to the latest version of the rRNA database SILVA (138.1; 99% ASVs from 515 F/806 R region of sequences) 93 . Based on an assessment of alpha rarefaction, a threshold of 6500 sequences/sample was established, retaining all samples for downstream analysis. A phylogenic tree was then constructed using the fragment-insertion plugin with SILVA at a p-sampling depth of the rarefaction threshold to impute high-quality reads and normalize for uneven sequencing depth between samples 94 . Alpha diversity (intra-community diversity) was measured using observed ASVs and the Phylogenetic diversity index. Additionally, the Shannon index was calculated for the subgroup and case-study analyses to capture richness and evenness at the species level. Beta diversity (inter-community diversity) was measured using Bray-Curtis dissimilarity.

For shotgun metagenomics, DNA was sequenced on the Illumina NextSeq 500 platform (Illumina, CA, USA) to generate 2 × 150 bp paired-end reads at greater sequencing depth with a minimum of 10 million reads. Raw Illumina sequencing reads underwent standard quality control with FastQC. Adapters were trimmed using TrimGalore. DNA sequences were aligned to Hg38 using bowtie2 95 . DNA sequences were then analyzed via the bio bakery pipeline 96 for taxonomic composition and potential functional content with MetaPhlAn4 and HUMAnN 3.0 (UniRef90 gene-families and MetaCyc metabolic pathways), using standard parameters. Functional profiling resulted in 8528 distinct Kyoto Encyclopedia of Genes and Genomes Orthology (KO) groups and 511 metabolic pathways, which align with previous human gut microbiome studies 96 .

Blood sample collection and biochemical analyses

All participants were tested between the hours of 6:00 a.m. and 9:00 a.m., after an overnight fast for body composition assessments (height, body weight, and total body composition) at weeks 0, 4, and 8. 12-h fasted venous blood samples (~20 mL) were collected into EDTA-coated vacutainer tubes and centrifuged (Hettich Rotina 46R5) for 15 min at 4000 ×  g at −4 °C. After separation, plasma was stored at −80 °C until analyzed. Undiluted plasma samples were sent to Eve Technologies (Calgary, Alberta, Canada) for assessment of inflammatory cytokines [Granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon-γ (IFNγ), interleukin (IL)-β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-23, and Tumor necrosis factor-α (TNFα)] using a high human sensitivity 14-plex cytokine assay (Millipore, Burlington, MA). Circulating LBP concentrations were quantified in duplicate using 1000x diluted plasma samples. A commercially available kit was used per the manufacturer’s protocol (Cat No. EH297RB, Thermo Fisher Scientific, Inc, Waltham, MA; intra-assay coefficient variation [CV] <10%).

Targeted plasma metabolomic analysis

For the plasma metabolomic analysis, a 12-h fasted venous blood sample (~20 mL) was collected into EDTA-coated vacutainer tubes and centrifuged (Hettich Rotina 46R5) for 15 min at 4000 ×  g at 4 °C. After separation, 2 mL of plasma was aliquoted and stored at −80 °C at the Biochemistry Laboratory at Skidmore College (Saratoga Springs, NY, USA). Samples were then sent to the Arizona Metabolomics Laboratory at ASU (Phoenix, AZ, USA) overnight on dry ice for analysis, where they were thawed at 4 °C and processed. Briefly, 50 μL of plasma from each sample was processed to precipitate proteins and extract metabolites by adding 500 μL MeOH and 50 μL internal standard solution (containing 1810.5 μM 13 C 3 -lactate and 142 μM 13 C 5 -glutamic acid). The mixture was vortexed (10 s) and stored for 30 min at –20 °C, then centrifuged at 224,000 ×  g for 10 min at 4 °C. Supernatants (450 μL) were extracted, transferred to new Eppendorf vials, and dried (CentriVap Concentrator; Labconco, Fort Scott, KS, USA). Samples were then reconstituted in 150 μL of 40% phosphate-buffered saline (PBS)/60% acetonitrile (ACN) and centrifuged again at 22,000 ×  g at 4 °C for 10 min. Supernatants (100 µL) were transferred to an LC autosampler vial for subsequent analysis. Quality control (QC) was performed by creating a pooled sample from all plasma samples and injecting once every ten experimental samples to monitor system performance.

The highly-reproducible targeted LC–MS/MS method used in the current investigation was modeled after previous studies 97 , 98 , 99 . The specific metabolites included in our targeted detection panel are representative of more than 35 biological pathways most essential to biological metabolism and have been successfully leveraged for the sensitive and broad detection of effects related to diet 100 , diseases 101 , drug treatment 102 , environmental contamination 103 , and lifestyle factors 104 . Briefly, LC–MS/MS experiments were performed on an Agilent 1290 UPLC-6490 QQQ-MS system (Santa Clara, CA, USA). Each sample was injected twice for analysis, 10 µL using negative and 4 µL using positive ionization modes. Chromatographic separations were performed in hydrophilic interaction chromatography (HILIC) mode on a Waters Xbridge BEH Amide column (150 × 2.1 mm, 2.5 µm particle size, Waters Corporation, Milford, MA, USA). The flow rate was 0.3 mL/min, the autosampler temperature was maintained at 4 °C, and the column compartment was set at 40 °C. The mobile phase system was composed of Solvents A (10 mM ammonium acetate, 10 mM ammonium hydroxide in 95% H 2 O/5% ACN) and B (10 mM ammonium acetate, 10 mM ammonium hydroxide in 95% ACN/5% H 2 O). After the initial 1 min isocratic elution of 90% Solvent B, the percentage of Solvent B decreased to 40% at t  = 11 min. The composition of Solvent B was maintained at 40% for 4 min ( t  = 15 min).

The mass spectrometer was equipped with an electrospray ionization (ESI) source. Targeted data acquisition was performed in multiple-reaction monitoring (MRM) mode. The LC–MS system was controlled by Agilent MassHunter Workstation software (Santa Clara, CA, USA), and extracted MRM peaks were integrated using Agilent MassHunter Quantitative Data Analysis software (Santa Clara, CA, USA).

GC–MS fecal short-chain fatty acid analysis

Before GC–MS analysis of SCFAs, frozen fecal samples were first thawed overnight under 4 °C. Then, 20 mg of each sample was homogenized with 5 μL hexanoic acid—6,6,6-d 3 (internal standard; 200 µM in H 2 O), 15 μL sodium hydroxide (NaOH [0.5 M]), and 500 μL MeOH. Samples were stored at −20 °C for 20 min and centrifuged at 22,000 ×  g for 10 min afterward. Next, 450 μL of supernatant was collected, and the sample pH was adjusted to 10 by adding 30 μL of NaOH:H 2 O (1:4, v-v). Samples were then dried, and the residues were initially derivatized with 40 µL of 20 mg/mL MeOX solution in pyridine under 60 °C for 90 min. Subsequently, 60 µL of MTBSTFA containing d 27 -mysristic acid was added, and the mixture was incubated at 60 °C for 30 min. The samples were then vortexed for 30 s and centrifuged at 22,000 ×  g for 10 min. Finally, 70 µL of supernatant was collected from each sample and injected into new glass vials for GC–MS analysis.

GC–MS conditions used here were adopted from a previously published protocol 105 . Briefly, GC–MS experiments were performed on an Agilent 7820 A GC-5977B MSD system (Santa Clara, CA); all samples were analyzed by injecting 1 µL of prepared samples. Helium was the carrier gas with a constant flow rate of 1.2 mL/min. Separation of metabolites was achieved using an Agilent HP-5 ms capillary column (30 m × 250 µm × 0.25 µm). Ramping parameters were as follows: column temperature was maintained at 60 °C for 1 min, increased at a rate of 10 °C/min to 325 °C, and then held at this temperature for 10 min. Mass spectral signals were recorded at an m/z range of 50–600, and data extraction was performed using Agilent Quantitative Analysis software. Following peak integration, metabolites were filtered for reliability. Only those with QC CV < 20% and a relative abundance of 1000 in > 80% of samples were retained for statistical analysis.

Untargeted fecal metabolomic analysis

Briefly, each fecal sample (~20 mg) was homogenized in 200 µL MeOH:PBS (4:1, v-v, containing 1810.5 μM 13 C 3 -lactate and 142 μM 13 C 5 -glutamic Acid) in an Eppendorf tube using a Bullet Blender homogenizer (Next Advance, Averill Park, NY). Then 800 µL MeOH:PBS (4:1, v-v, containing 1810.5 μM 13 C 3 -lactate and 142 μM 13 C 5 -glutamic Acid) was added, and after vortexing for 10 s, the samples were stored at −20 °C for 30 min. The samples were then sonicated in an ice bath for 30 min. The samples were centrifuged at 22,000 ×  g for 10 min (4 °C), and 800 µL supernatant was transferred to a new Eppendorf tube. The samples were then dried under vacuum using a CentriVap Concentrator (Labconco, Fort Scott, KS). Prior to MS analysis, the obtained residue was reconstituted in 150 μL 40% PBS/60% ACN. A quality control (QC) sample was pooled from all the study samples.

The untargeted LC–MS metabolomics method used here was modeled after that developed and used in a growing number of studies 106 , 107 , 108 . Briefly, all LC–MS experiments were performed on a Thermo Vanquish UPLC-Exploris 240 Orbitrap MS instrument (Waltham, MA). Each sample was injected twice, 10 µL for analysis using negative ionization mode and 4 µL for analysis using positive ionization mode. Both chromatographic separations were performed in hydrophilic interaction chromatography (HILIC) mode on a Waters XBridge BEH Amide column (150 × 2.1 mm, 2.5 µm particle size, Waters Corporation, Milford, MA). The flow rate was 0.3 mL/min, autosampler temperature was kept at 4 °C, and the column compartment was set at 40 °C. The mobile phase was composed of Solvents A (10 mM ammonium acetate, 10 mM ammonium hydroxide in 95% H 2 O/5% ACN) and B (10 mM ammonium acetate, 10 mM ammonium hydroxide in 95% ACN/5% H 2 O). After the initial 1 min isocratic elution of 90% B, the percentage of Solvent B decreased to 40% at t  = 11 min. The composition of Solvent B maintained at 40% for 4 min ( t  = 15 min), and then the percentage of B gradually went back to 90%, to prepare for the next injection. Using mass spectrometer equipped with an electrospray ionization (ESI) source, we collected untargeted data from 70 to 1050 m/z.

To identify peaks from the MS spectra, we made extensive use of the in-house chemical standards (~600 aqueous metabolites), and in addition, we searched the resulting MS spectra against the HMDB library, Lipidmap database, METLIN database, as well as commercial databases including mzCloud, Metabolika, and ChemSpider. The absolute intensity threshold for the MS data extraction was 1000, and the mass accuracy limit was set to 5 ppm. Identifications and annotations used available data for retention time (RT), exact mass (MS), MS/MS fragmentation pattern, and isotopic pattern. We used the Thermo Compound Discoverer 3.3 software for aqueous metabolomics data processing. The untargeted data were processed by the software for peak picking, alignment, and normalization. To improve rigor, only the signals/peaks with CV < 20% across quality control (QC) pools, and the signals showing up in >80% of all the samples were included for further analysis. To ensure the robustness of our model validation, we employed an enhanced validation approach by repeating the LOOCV process 100 times. Each iteration involves excluding one sample from the dataset to serve as the test set, with the model being trained on the remaining samples. This approach, referred to as ‘repeated LOOCV’, was adopted to mitigate bias and provide a thorough validation of our model’s predictive capability. The method signifies the number of repetitions of the LOOCV process, rather than splitting the dataset into 100 equal parts.

Multi-omics data analysis

For MOFA, bacterial 16S rRNA ASVs and plasma metabolites were integrated using the MOFA2 package 55 . Before integration, ASV sequences were filtered (minimum of 5 ASV in greater than 10% of all samples), collapsed to the genus level, and scaled using a centralized-log-ratio, as described previously 109 . Plasma metabolites were scaled and normalized as described in the metabolome analysis. The inputs for MOFA model training comprised 53 taxa and 138 metabolites. The latent factors and feature loadings were extracted from the best-trained model with the built-in functions of MOFA2. After model fitting, the number of factors was estimated by requiring a minimum of 2% variance explained across all microbiome modalities.

Integrating microbial taxa with the same filtration as stated above (at the genus level from 16S amplicon sequencing and species level from metagenomic sequencing) and cytokine data and fecal metabolomic data, respectively, was conducted with GFLASSO (R package: GFLASSO, v0.0.0.9000). This correlation-based network solution can handle multiple response variables for a given set of predictors (in this case: 1. cytokine abundances predicted by microbial taxa response; and 2. fecal metabolite response predicted by microbial taxa). Solution parsimony was determined by an unweighted (i.e., presence or absence of association by imposing a correlation threshold) network structure. The regularization and fusion parameters were determined from the smallest root mean squared error (RMSE) estimate via cross-validation, accounting for interdependencies among microbial features. The tested parameters encompassed all combinations between λ and γ with values ranging from 0 to 1 (inclusive) in step increments of 0.1. GFLASSO coefficient matrices were constructed using a threshold coefficient of >0.02 to discern the strongest associative signals.

Statistical analysis

Gastrointestinal symptom scores were on the low end of the GSRS scale and not normally distributed; therefore, nonparametric statistical tests were applied. Symptom prevalence (number of scores ≥ 2) and moderate symptom prevalence (≥4) for total, upper, and lower GI GSRS clusters were analyzed using contingency tables. Specifically, differences between IF-P and CR GI symptoms at baseline were compared using a Fisher’s Exact test, whereas baseline vs. weeks four and eight values were compared with McNemar’s test. Stool weight, BSS, fecal pH, plasma cytokines and LBP, and SCFAs were assessed for normality with Q-Q plots and Shapiro-Wilk tests and log-transformed where appropriate. These were then tested for time and interaction (group × time) effects using linear-mixed effect (LME) models, with each participant included as a random effect.

For analysis and visualization of the microbiome data, artifacts generated in QIIME2 were imported into the R environment (v4.2.2) using the phyloseq package (v1.42.0) 110 . Before conducting downstream analyses, sequences were filtered to remove all non-bacterial sequences, including archaea, mitochondria, and chloroplasts. After assessing normality (Shapiro-Wilk’s tests), LME models were used to test the effect of time and the interaction of group and time with the covariates of age and sex with each participant included as a random effect on the alpha diversity metrics using the nLME package (v3.1.160). For beta diversity, a nested permutational analysis of variance (PERMANOVA) was conducted on Bray-Curtis dissimilarities using the Adonis test in the vegan package (v2.6.2) with 999 permutations. The PERMANOVA model incorporated the factors of time, individual, interaction (group × time), and participant (nested factor). A permutation test for homogeneity in multivariate dispersion (PERMDISP) was conducted using the ‘betadisper’ function in the vegan package to compare dispersion. To support the Adonis analysis, intra-individual differences were also compared between groups, as previously described 111 , by calculating the within-subject distance for paired samples (baseline vs. weeks four and eight) and testing for group distances (Wilcoxon rank-sum test). Differential abundance analysis was performed using MaAsLin2 (v1.12.0) 18 . To detect changes in microbial features between groups over time, we built linear-mixed models that include group, time, and their interaction, with age and sex as covariates and the participant as a random factor. Before analysis, raw counts from the ASV table were filtered for any sequence not present five times in at least 30% of all samples. A significant p-value for the product term indicates that changes in microbial features differed over time between groups. The Benjamini–Hochberg (BH) procedure was used to correct for multiple testing at ≤0.10. To assess the correlation between changes in specific taxa and biomarkers over the eight-week intervention, Spearman correlation tests were performed.

Univariate and multivariate analyses of plasma metabolites and metabolic ontology analysis were performed, and results were visualized using the MetaboAnalystR 5.0 112 . Human metabolomic data were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) human pathway library to analyze predicted states 113 . The data were log 10 -transformed, and Pareto scaled to approximate normality before all analyses. A GLM was constructed with age, sex, and time as covariates to determine significantly affected metabolites by group intervention. Levene’s test was performed to detect significant homogeneity. The BH procedure was used to correct for multiple testing at ≤0.10. Fecal metabolomic analysis for the subgroup comparison was performed by assessing logFC values between groups with a Wilcoxon rank-sum test with BH adjustment. For pathway analysis, the impact was calculated using a hypergeometric test, while significance was determined using a test of relative betweenness centrality. Importantly, the BH procedure was not applied to pathway and enzyme enrichment analyses for the subgroup assessment since these analyses involve testing the significance of multiple related hypotheses rather than independent hypotheses, which is too conservative, resulting in false negative results.

For MOFA, latent factors explaining ≥2.0% of model variance from the plasma metabolomic and amplicon microbiome data were used to perform Spearman correlations on anthropometric and nutritional data and compared between IF-P and CR groups using Wilcoxon rank-sum tests. The highest beta coefficients (>0.3) detected from GFLASSO models were further assessed by performing Spearman correlations of select microbial features with the response variables (i.e., cytokines and fecal metabolites). All statistical tests were performed with a significance level of p  < 0.05 and BH correction of p .adj < 0.10. In addition, we present data in this study in accordance with the ‘Strengthening The Organization and Reporting of Microbiome Studies’ (STORMS) guidelines for human microbiome research 114 .

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

The microbiome sequencing data generated in this study have been deposited in the BioProject Database of National Centre for Biotechnology Information database under accession code PRJNA847971 . The metadata data linking the microbiome sequences with the appropriate sample ID and intervention in this study are provided in Supplementary Data  1 . The processed data are available at https://github.com/Alex-E-Mohr/GM-Remodeling-IF-ProteinPacing-vs-CaloricRestriction .  Source data are provided with this paper.

Code availability

The R code used for analysis and figure generation for reproducibility purposes are available at: https://github.com/Alex-E-Mohr/GM-Remodeling-IF-ProteinPacing-vs-CaloricRestriction . 115

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Acknowledgements

We thank the trial volunteers for their dedication and commitment to the study protocol. We are grateful for the research assistants from Skidmore College who provided valuable assistance with study protocol design, scheduling, recruitment, data testing, collection, entry, and statistical analysis, and preparation of manuscripts: Molly Boyce, Jenny Zhang, Melissa Haas, Olivia Furlong, Emma Valdez, Jessica Centore, Annika Smith, Kaitlyn Judd, Aaliyah Yarde, Katy Ehnstrom, Dakembay Hoyte, Sheriden Beard, Heather Mak, and Monique Dudar. We are grateful for the extensive guidance and counseling provided by the registered dietitian Jaime Martin. We thank research coordinator Michelle Poe for her superior dedication to all aspects of the study. This study was primarily funded by an unrestricted grant from Isagenix International LLC to P.J.A. (grant #:1911-859), with secondary funding provided to K.L.S.

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Contributions

Study conceived and designed: P.J.A. Manuscript preparation with input from all authors: A.E.M., K.L.S., D.A.B., P.J., C.M.W., D.D.S., R.K.-B., H.G., J.K.-S., K.M.A., E.G., and P.J.A. Randomized study design and execution: K.M.A., and P.J.A. Microbiome analysis: A.E.M., D.A.B., C.M.W., and R.K.-B. Blood analyte analysis: A.E.M., K.L.S., and P.J.A. Metabolomic analysis: A.E.M., Y.J., H.G., and P.J. Statistical analysis and data presentation: A.E.M., C.M.W., D.D.S., R.K.-B., and P.J.A. Supervision and funding: K.L.S., E.G., and P.J.A.

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Competing interests.

P.J.A. is a consultant for Isagenix International LLC, the study’s sponsor, he is an advisory board member of the International Protein Board (iPB), and he receives financial compensation for books and keynote presentations on protein pacing ( www.paularciero.com ). Eric Gumpricht is employed by Isagenix International, LLC, the funding source for this research. Isagenix International, LLC had no role in the study design, data collection, analysis, or decision to publish. No authors have financial interests regarding the outcomes of this investigation. The other authors declare no competing interests.

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Mohr, A.E., Sweazea, K.L., Bowes, D.A. et al. Gut microbiome remodeling and metabolomic profile improves in response to protein pacing with intermittent fasting versus continuous caloric restriction. Nat Commun 15 , 4155 (2024). https://doi.org/10.1038/s41467-024-48355-5

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DOI : https://doi.org/10.1038/s41467-024-48355-5

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