Muno-IgY Supplementation Improves Respiratory Health, Immune Response, and Exercise-Induced Physiological Stress in Healthy Adults: A Randomized Controlled Pilot Study
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Thank you for the opportunity to review this manuscript. While the study addresses a timely and potentially interesting topic and employs a randomized, double-blind, placebo-controlled design, the manuscript in its current form does not meet the standards required for publication. The concerns outlined below collectively indicate that the manuscript does not yet meet the standards of a high-quality review article and is not suitable for publication in its current form.
The primary concern is systematic over interpretation of exploratory findings, particularly in the Discussion and Conclusions. Across multiple domains, including URTI outcomes, immune and inflammatory biomarkers, exercise-induced responses, and gut microbiome analyses, the manuscript repeatedly emphasizes directional trends, mechanistic plausibility, and implied biological effects despite limited statistical support and an underpowered pilot design. Causal and stabilizing language is frequently used where only descriptive or hypothesis-generating interpretations are warranted.
Microbiome analyses are of particular concern. Species-level and post-hoc subgroup analyses are extensively described despite the absence of formal statistical inference and extremely small sample sizes in key subgroups. These findings are discussed using functional language that implies biological relevance (e.g., stabilization, modulation) not supported by the data.
Although limitations are acknowledged, they are not adequately integrated into the interpretation, resulting in conclusions that exceed the evidentiary strength of the study. Safety conclusions are also overgeneralized given the short duration, small sample size, and healthy study population.
Addressing these issues would require substantial restructuring, condensation, and reframing of the manuscript, particularly the Discussion and Conclusions, to align claims with the exploratory nature of the data.
Abstract: The conclusions somewhat exceed the results by implying broader health benefits (e.g., respiratory health) that were not statistically established. The conclusions tend to overinterpret non-significant and exploratory findings. Numerical differences without statistical significance should be described more cautiously, and causal language should be tempered to reflect the pilot and hypothesis-generating nature of the study. Greater alignment between the reported results and the strength of the conclusions is recommended.
Line 93: The study aims are exceptionally broad for a pilot trial, encompassing clinical outcomes (URTI incidence, duration, and severity), multiple biomarker domains (inflammatory, immune, muscle damage), gut microbiome modulation, quality of life, tolerance, and safety. This breadth risks overextending the study’s inferential capacity and may dilute focus. It is recommended to clearly distinguish primary, secondary, and exploratory outcomes, which is particularly important given the wide range of endpoints assessed.
The authors repeatedly use interpretive and directional language (e.g., “associated with,” “suggesting potential modulation,” “consistent and reproducible enrichment,” “stabilizing effect”). Such phrasing implies biological or mechanistic effects that are not supported in the absence of statistical testing, particularly given the small sample size.
Phrasing such as “investigates the efficacy” and “evaluates its capacity to buffer” suggests demonstrated effectiveness, which is premature for a pilot study. More cautious wording (e.g., “explores potential effects” or “assesses feasibility and preliminary signals”) would be more appropriate.
The use of mean ± SEM for descriptive baseline data may underrepresent variability in this small pilot study, and reporting mean ± SD would be more appropriate. The high female predominance and small subgroup sizes should be acknowledged as limitations to generalizability.
Line 555: The URTI-restricted microbiome analysis (n = 2 in the Muno-IgY group) is severely underpowered and highly vulnerable to outlier effects. Despite this, the manuscript draws comparatively strong conclusions regarding “consistent” enrichment and “stabilizing effects,” which are not justified and risk misleading readers.
Line 521: References to “opportunistic or potentially pathogenic species,” “butyrate-producing taxa,” suggest functional or clinical implications that are not directly assessed in this study and should therefore be avoided or clearly contextualized.
Line 101 & 568: In “This randomized, double-blind, placebo-controlled pilot study provides an integrated assessment of the effects of Muno-IgY… “ “….provides an integrated assessment of the effects” implies demonstrated efficacy and it is suggested to be softened to “…provides an exploratory assessment of the potential effects of Muno-IgY..”
Line 574: “Participants receiving Muno-IgY experienced a numerically lower incidence of URTIs…” Acceptable descriptively, but becomes problematic when followed by efficacy framing and it is suggested to be softened to “A numerically lower URTI incidence was observed in the Muno-IgY group, though differences were not statistically significant.”
Line 576: “The approximately 60% relative reduction in URTI incidence aligns with prior studies…” percent reduction is emphasized despite non-significance and low event counts and it is suggested to be softened to “Although a numerically lower URTI incidence was observed, the small number of events and lack of statistical significance limit meaningful comparison with prior studies.”
Line 583: “The observed trends are biologically plausible given IgY’s known mechanisms of action…” mechanistic plausibility is used to compensate for weak evidence and it is suggested to be softened to “While mechanistic hypotheses exist, the present data do not allow confirmation of biological effects.”
Line 586: “Muno-IgY may provide broader mucosal protection…”broad clinical implication unsupported by data and it is suggested to be softened to “Muno-IgY is hypothesized to have mucosal activity, which warrants further investigation”
Line 596: “The observed modulation of LBP suggests a potential role for Muno-IgY in supporting gut barrier integrity…” single biomarker change interpreted as a functional gut barrier effect and it is suggested to be softened to “Changes in LBP should be interpreted cautiously and cannot be used to infer gut barrier function in this pilot study
Line 641: “Exploratory analyses revealed consistent directional shifts in microbial composition…” “Consistent” implies reproducibility not supported by statistics and it is suggested to be softened to “Exploratory analyses showed descriptive differences in microbial composition.
Line 662” “These findings suggest a stabilizing effect of Muno-IgY on the gut microbiome…” “Stabilizing” is a strong functional claim and it is suggested to be softened to “These observations are descriptive and hypothesis-generating.
“Potentially enhancing short-chain fatty acid production…” SCFAs were not measured. Please remove entirely or explicitly state that SCFAs were not assessed.
Line 685: “Overall, these findings suggest that Muno-IgY may help support immune resilience…” cumulative overstatement despite exploratory design and it is suggested to be softened to “Overall, the findings are exploratory and support further investigation in larger, adequately powered trials.”
Author Response
Thank you very much for taking the time to review this manuscript. Please find detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.
Reviewer 1
Thank you for the opportunity to review this manuscript. While the study addresses a timely and potentially interesting topic and employs a randomized, double-blind, placebo-controlled design, the manuscript in its current form does not meet the standards required for publication. The concerns outlined below collectively indicate that the manuscript does not yet meet the standards of a high-quality review article and is not suitable for publication in its current form.
The primary concern is systematic over interpretation of exploratory findings, particularly in the Discussion and Conclusions. Across multiple domains, including URTI outcomes, immune and inflammatory biomarkers, exercise-induced responses, and gut microbiome analyses, the manuscript repeatedly emphasizes directional trends, mechanistic plausibility, and implied biological effects despite limited statistical support and an underpowered pilot design. Causal and stabilizing language is frequently used where only descriptive or hypothesis-generating interpretations are warranted.
Microbiome analyses are of particular concern. Species-level and post-hoc subgroup analyses are extensively described despite the absence of formal statistical inference and extremely small sample sizes in key subgroups. These findings are discussed using functional language that implies biological relevance (e.g., stabilization, modulation) not supported by the data.
Although limitations are acknowledged, they are not adequately integrated into the interpretation, resulting in conclusions that exceed the evidentiary strength of the study. Safety conclusions are also overgeneralized given the short duration, small sample size, and healthy study population.
Addressing these issues would require substantial restructuring, condensation, and reframing of the manuscript, particularly the Discussion and Conclusions, to align claims with the exploratory nature of the data.
Comment 1: Abstract: The conclusions somewhat exceed the results by implying broader health benefits (e.g., respiratory health) that were not statistically established. The conclusions tend to overinterpret non-significant and exploratory findings. Numerical differences without statistical significance should be described more cautiously, and causal language should be tempered to reflect the pilot and hypothesis-generating nature of the study. Greater alignment between the reported results and the strength of the conclusions is recommended.
We thank the reviewer for the comment. The abstract results and conclusions have been revised to better align with the results and the exploratory nature of the study (Page 1, Lines 27-39).
Comment 2: Line 93: The study aims are exceptionally broad for a pilot trial, encompassing clinical outcomes (URTI incidence, duration, and severity), multiple biomarker domains (inflammatory, immune, muscle damage), gut microbiome modulation, quality of life, tolerance, and safety. This breadth risks overextending the study’s inferential capacity and may dilute focus. It is recommended to clearly distinguish primary, secondary, and exploratory outcomes, which is particularly important given the wide range of endpoints assessed. Phrasing such as “investigates the efficacy” and “evaluates its capacity to buffer” suggests demonstrated effectiveness, which is premature for a pilot study. More cautious wording (e.g., “explores potential effects” or “assesses feasibility and preliminary signals”) would be more appropriate.
Thank you for pointing it out. In response, we have revised the introduction to clearly distinguish the primary, secondary, and exploratory aims of this pilot study. The text now emphasizes that the study is exploratory and hypothesis-generating (Page 3, Lines 97-106).
Comment 3: The authors repeatedly use interpretive and directional language (e.g., “associated with,” “suggesting potential modulation,” “consistent and reproducible enrichment,” “stabilizing effect”). Such phrasing implies biological or mechanistic effects that are not supported in the absence of statistical testing, particularly given the small sample size.
Thank you for bringing this to our attention. Changes have been made in the manuscript to address the concern raised by the reviewer (Page 19, Lines 539-544; Page 20, Lines 549-554; Page 23, Lines 640-641; Page 23, Lines 662-665; Page 23, Lines 675-676)
Comment 4: The use of mean ± SEM for descriptive baseline data may underrepresent variability in this small pilot study, and reporting mean ± SD would be more appropriate. The high female predominance and small subgroup sizes should be acknowledged as limitations to generalizability.
Thank you for this comment. We agree that reporting mean ± SD is more appropriate for descriptive baseline data in a small pilot study. Accordingly, baseline data have been revised to be presented as mean ± SD throughout the manuscript. In addition, we have explicitly acknowledged the high female predominance and the small subgroup sizes as limitations that may restrict the generalizability of the findings, and this has been added to the Limitations section of the Discussion.
Comment 5: Line 555: The URTI-restricted microbiome analysis (n = 2 in the Muno-IgY group) is severely underpowered and highly vulnerable to outlier effects. Despite this, the manuscript draws comparatively strong conclusions regarding “consistent” enrichment and “stabilizing effects,” which are not justified and risk misleading readers.
We thank the reviewer for this important observation and agree with it. We have revised the manuscript to temper the language and interpretation of these findings substantially (Page 22, Lines 575-579, Lines 590-595; Page 25, Lines 703-704).
Comment 6: Line 521: References to “opportunistic or potentially pathogenic species,” “butyrate-producing taxa,” suggest functional or clinical implications that are not directly assessed in this study and should therefore be avoided or clearly contextualized.
We agree with the reviewer that references to functional or clinical characteristics inferred from taxonomic annotations may imply effects that were not directly measured in this study. The manuscript has been revised to clearly contextualize these descriptors as literature-based classifications rather than assessed functional outcomes (Page 20, Line 549-559).
Comment 7: Line 101 & 568: In “This randomized, double-blind, placebo-controlled pilot study provides an integrated assessment of the effects of Muno-IgY… “ “….provides an integrated assessment of the effects” implies demonstrated efficacy and it is suggested to be softened to “…provides an exploratory assessment of the potential effects of Muno-IgY..”
We thank the reviewer for this suggestion and agree that the original phrasing may imply demonstrated efficacy, which is not appropriate for a pilot study. Accordingly, the text has been revised (Page 3, Line 97-106; Page 23, Line 601)
Comment 8: Line 574: “Participants receiving Muno-IgY experienced a numerically lower incidence of URTIs…” Acceptable descriptively, but becomes problematic when followed by efficacy framing and it is suggested to be softened to “A numerically lower URTI incidence was observed in the Muno-IgY group, though differences were not statistically significant.”
We thank the reviewer for this important observation and agree with it. We have revised the manuscript accordingly (Page 23, Line 611-613).
Comment 9: Line 576: “The approximately 60% relative reduction in URTI incidence aligns with prior studies…” percent reduction is emphasized despite non-significance and low event counts and it is suggested to be softened to “Although a numerically lower URTI incidence was observed, the small number of events and lack of statistical significance limit meaningful comparison with prior studies.”
We thank the reviewer for this important observation and agree with it. We have revised the manuscript accordingly (Page 23, Line 613-617).
Comment 10: Line 583: “The observed trends are biologically plausible given IgY’s known mechanisms of action…” mechanistic plausibility is used to compensate for weak evidence and it is suggested to be softened to “While mechanistic hypotheses exist, the present data do not allow confirmation of biological effects.”
We thank the reviewer for this important observation and agree with it. We have revised the manuscript accordingly (Page 23, Line 619-622).
Comment 11: Line 586: “Muno-IgY may provide broader mucosal protection…”broad clinical implication unsupported by data and it is suggested to be softened to “Muno-IgY is hypothesized to have mucosal activity, which warrants further investigation.
We thank the reviewer for this important observation and agree with it. We have revised the manuscript accordingly (Page 23, Line 622-624).
Comment 12: Line 596: “The observed modulation of LBP suggests a potential role for Muno-IgY in supporting gut barrier integrity…” single biomarker change interpreted as a functional gut barrier effect and it is suggested to be softened to “Changes in LBP should be interpreted cautiously and cannot be used to infer gut barrier function in this pilot study
We thank the reviewer for this important observation and agree with it. We have revised the manuscript accordingly (Page 24, Line 636-638).
Comment 13: Line 641: “Exploratory analyses revealed consistent directional shifts in microbial composition…” “Consistent” implies reproducibility not supported by statistics and it is suggested to be softened to “Exploratory analyses showed descriptive differences in microbial composition.
We thank the reviewer for this important observation and agree with it. We have revised the manuscript accordingly (Page 24, Line 676-677).
Comment 14: Line 662” “These findings suggest a stabilizing effect of Muno-IgY on the gut microbiome…” “Stabilizing” is a strong functional claim and it is suggested to be softened to “These observations are descriptive and hypothesis-generating.
We thank the reviewer for this important observation and agree with it. We have revised the manuscript accordingly (Page 24, Line 702-704).
Comment 15: “Potentially enhancing short-chain fatty acid production…” SCFAs were not measured. Please remove entirely or explicitly state that SCFAs were not assessed.
The sentence has been removed as per the suggestion (Page 25, Lines 703-704).
Comment 16: Line 685: “Overall, these findings suggest that Muno-IgY may help support immune resilience…” cumulative overstatement despite exploratory design and it is suggested to be softened to “Overall, the findings are exploratory and support further investigation in larger, adequately powered trials.”
We thank the reviewer for this important observation and agree with it. We have revised the manuscript accordingly (Page 24, Line 702-704).
Reviewer 2 Report
Comments and Suggestions for Authors
The only observations I would like to raise are: 1) importante to describe the techniques used to measure serum IgA levels and inflammatory biomarkers; 2) The resolution of the figures should be improved by increasing the size of numbers and letters, particularly in Figures 8–12.
And, the importance of future larger-scale clinical trials in populations at increased risk of respiratory infections or physiological stress, as well as studies to elucidate the underlying mechanisms of action of Muno-IgY.
Author Response
Thank you very much for taking the time to review this manuscript. Please find detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.
Comment 1: It is important to describe the techniques used to measure serum IgA levels and inflammatory biomarkers.
Thank you for pointing it out. The details of the assay have been updated on page 6, lines 223-225, 242-252, and page 7, Lines 265-277.
Comment 2: The resolution of the figures should be improved by increasing the size of numbers and letters, particularly in Figures 8–12.
We thank the reviewer for this comment. The reduced readability of text and numerical labels in Figures 8–12 appears to be due to resolution loss during insertion of the figures into the manuscript document. High-resolution, publication-quality versions of all figures (including Figures 8–12) were uploaded separately as a compressed ZIP folder at the time of submission.
Comment 3: And, the importance of future larger-scale clinical trials in populations at increased risk of respiratory infections or physiological stress, as well as studies to elucidate the underlying mechanisms of action of Muno-IgY.
We thank the reviewer for this comment. We have revised the manuscript accordingly (Page 25, Line 723-725).
Reviewer 3 Report
Comments and Suggestions for Authors
I have reviewed the manuscript entitled "Muno-IgY Supplementation Improves Respiratory Health, Immune Response, and Exercise-Induced Physiological Stress in Healthy Adults: A Randomized Controlled Pilot Study" by Fathima S. et al.
The manuscript is novel and covers a topic with significant clinical interest. The methodological approach is generally acceptable, though some issues remain to be resolved. The statistical tools used are appropriate. The text is well organized. The results are properly presented. All Tables and Figures are informative and easy-to-study. The discussion is coherent and the conclusion are largely supported by the results.
The manuscript, if revised properly, could deserve publication. However, the authors are ecouraged to assess the following issues in an effort to further clarify and strengthen the validity of the results.
Major issues:
1) Lines 26-27: The authors report that "URTI incidence was numerically lower in the Muno-IgY group compared with placebo (14.3% vs. 35.7%), with shorter average duration and fewer missed workdays (p > 0.05).". Since the result is non-significant, "numerical" comparisons can be misinterpreted. The authors are kindly suggested to appropriately revise the phrase so as to explicitly indicate non-significance. In detail, they are kindly suggested to define the exact definition of “history of URTI” (time window; frequency threshold; physician diagnosis vs self-report; whether allergies were excluded), explain how URTI history was verified by each participant (medical record, ICD codes, diaries, rapid tests, etc.), and assess baseline comparability of URTI history severity/frequency by arm.
2) Lines 108-109: The authors state that "Healthy adults who had self-reported at least one URTI in the previous six months were eligible for enrollment". However, the use of self-reported disease status as an inclusion criterion might cause systematic selection and misclassification biases that directly threaten internal validity.
3) Line 681-682" The auhtors report that "Limitations include the small sample size". Though there is no evidence of an a priori power justification, considering the pilot nature of the present study, analyses should be interpreted as exploratory and susceptible to both Type I and Type II error. This should be explicitly explained and discusse in the revised text.
MInor issues
1) Line 20: It seems that something is missing in text "Methods: in this 12-week". Please confirm/revise appropriately..
Author Response
Thank you very much for taking the time to review this manuscript. Please find detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.
Comment 1: Lines 26-27: The authors report that "URTI incidence was numerically lower in the Muno-IgY group compared with placebo (14.3% vs. 35.7%), with shorter average duration and fewer missed workdays (p > 0.05).". Since the result is non-significant, "numerical" comparisons can be misinterpreted. The authors are kindly suggested to appropriately revise the phrase so as to explicitly indicate non-significance. In detail, they are kindly suggested to define the exact definition of “history of URTI” (time window; frequency threshold; physician diagnosis vs self-report; whether allergies were excluded), explain how URTI history was verified by each participant (medical record, ICD codes, diaries, rapid tests, etc.), and assess baseline comparability of URTI history severity/frequency by arm.
Page 1, Line 27-29: We thank the reviewer for this comment. We have revised the text in the abstract to clarify that the differences were not statistically significant. The updated sentence now reads:
“URTI incidence was lower in the Muno-IgY group compared with placebo (14.3% vs. 35.7%), with shorter average duration and fewer missed workdays, though differences were not statistically significant (p > 0.05).”
Comment 2: In detail, they are kindly suggested to define the exact definition of “history of URTI” (time window; frequency threshold; physician diagnosis vs self-report; whether allergies were excluded), explain how URTI history was verified by each participant (medical record, ICD codes, diaries, rapid tests, etc.), and assess baseline comparability of URTI history severity/frequency by arm.
We thank the reviewer for the request for clarification. In the manuscript, we have defined how URTIs were assessed during the study (Page 6, Lines 206-208; Page 12, Lines 388-392). Participants self-reported URTI symptoms daily in study diaries throughout the 12-week intervention. Reported symptoms were subsequently evaluated by the Qualified Investigator (QI) to confirm a clinical diagnosis, ensuring that events reflected true URTIs rather than allergies or unrelated symptoms. The duration and severity of URTIs were calculated as described in Section 2.3.1 and 4.4.1 (Page 12, Lines 397-401; Lines 414-416), and baseline comparability was considered by reporting the incidence per study group. Due to the small number of events, no formal statistical comparison of baseline URTI history severity/frequency was performed (Page 7-8, Lines 287-292); however, demographics and baseline characteristics were balanced between study arms (Table 2).
Comment 3: Lines 108-109: The authors state that "Healthy adults who had self-reported at least one URTI in the previous six months were eligible for enrollment". However, the use of self-reported disease status as an inclusion criterion might cause systematic selection and misclassification biases that directly threaten internal validity.
Thank you for highlighting this point. Eligibility based on self-reported URTI could introduce misclassification or selection bias. However, the study population was confirmed to be generally healthy at screening, and all reported URTI episodes during the study were prospectively documented and reviewed by the Qualified Investigator to confirm a clinical diagnosis. While self-report may have limitations, this approach is commonly used in clinical studies of healthy adults and was necessary to identify participants with a history of URTIs. We have added a statement to the Discussion section acknowledging this as a potential limitation regarding internal validity (Page 24, Lines 718-725).
Comment 4: Line 681-682" The authors report that "Limitations include the small sample size". Though there is no evidence of an a priori power justification, considering the pilot nature of the present study, analyses should be interpreted as exploratory and susceptible to both Type I and Type II error. This should be explicitly explained and discussed in the revised text.
We thank the reviewer for highlighting this point. We have revised the manuscript to explicitly acknowledge that, as a pilot study without formal power calculations, all analyses are exploratory and susceptible to both Type I and Type II errors. The revised text is included in the Discussion section (Page 25, Lines 737-743).
Minor issues
Comment 5: Line 20: It seems that something is missing in text "Methods: in this 12-week". Please confirm/revise appropriately..
We thank the reviewer for the comment. The sentence (Page 1, Lines 21-23) has been revised for clarity to read: 'In this 12-week, double-blind, placebo-controlled trial, 28 healthy adults with a history of URTI were randomly assigned to receive Muno-IgY or placebo, and URTI incidence, duration, and severity were recorded daily.'
Round 2
Reviewer 3 Report
Comments and Suggestions for Authors
I have reviewed the revised version of the manuscript entitled "Muno-IgY Supplementation Improves Respiratory Health, Immune Response, and Exercise-Induced Physiological Stress in Healthy Adults: A Randomized Controlled Pilot Study".
The authors have adequately responded to every query raised. The revised manuscript provide all necessary alterations and corrections to fulfill the issues opened during the review process. The quality of the manuscript has been improved. There are no additional comments.
