Review Reports - Enhancing Exercise Performance Under Hypoxia: A Network Meta-Analysis and Animal Experimental Validation of Plant Bioactive Compounds

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor,

Thank you for the invitation to review the manuscript entitled “Enhancing Exercise Performance under Hypoxia: A Network Meta-Analysis and Animal Experimental Validation of Plant Bioactive Compounds.” I appreciate the opportunity to evaluate this work. The study addresses an interesting topic by combining a network meta-analysis with experimental validation in an animal model. However, several methodological and reporting issues need to be clarified before the manuscript can be considered for publication. I have provided detailed comments and suggestions to help improve the manuscript.

Major revisions

Comment 1: The search strategy and eligibility criteria require clarification. Although the authors report that PubMed, Web of Science, and CNKI were searched, the strategy appears limited and potentially biased. For example, the search terms focus mainly on “hypoxia” and “alleviation,” which may not capture all relevant literature on exercise performance under hypoxic conditions. Furthermore, the restriction to publications from 2019–2024 should be justified. The complete strategy should be reported more clearly in the main text rather than only in the appendix.

Comment 2: The methodology of the network meta-analysis requires clearer reporting. The authors state that a frequentist framework using the MVMETA command in Stata was used, but several methodological aspects are not sufficiently described, including: Handling of multi-arm studies, Assessment of transitivity assumptions, Methods used to evaluate heterogeneity, Assessment of publication bias beyond visual funnel plots, These issues are critical for interpreting the robustness of the ranking results.

Comment 3: The manuscript reports that the SYRCLE tool was used to assess risk of bias. However: Detailed results are not sufficiently presented, it is unclear how the scoring system (1–3) influenced the analysis, No sensitivity analysis excluding high-risk studies is provided. Given that 10% of studies were classified as high risk, this issue should be addressed more thoroughly.

Comment 4: Justification of compound combinations in the animal experiment. The rationale for selecting the two plant compound combinations is insufficiently explained. For example, Intervention 1 includes astragaloside IV, ginsenoside Rg1, puerarin, and vitamin D3, but the manuscript does not clearly describe the criteria used for combining these compounds following the network meta-analysis ranking. A clearer explanation of the decision process is necessary.

Comment 5: Several methodological aspects of the animal experiment should be clarified: The sample size is small (n = 6 per group), and no power calculation is reported. The duration of the intervention may be insufficient to fully assess long-term adaptations to hypoxia. It is unclear whether investigators were blinded during outcome measurements. These limitations should be discussed more explicitly.

Comment 6: The statistical approach used in the animal experiments requires clarification. The authors report using one-way ANOVA with Duncan’s multiple range test, but it is unclear whether: assumptions of normality and homoscedasticity were tested, corrections for multiple comparisons were applied, effect sizes were calculated. More detailed statistical reporting would strengthen the results.

Comment 7: The conclusions sometimes overstate the findings. For example, the manuscript suggests that plant bioactive compounds “improve exercise endurance and performance under hypoxic conditions”, while the evidence is limited to a short-term mouse model. These conclusions should be toned down and framed more cautiously.

Minor revisions

Some abbreviations appear without prior definition (e.g., PAH, GP, ASF).
Figures showing network plots and forest plots could be improved for readability.
Language editing is recommended to improve clarity in several sections of the manuscript.
The discussion could better integrate previous literature on nutraceutical approaches to hypoxia adaptation

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents an innovative approach by combining network meta-analysis with experimental validation in hypoxic mice, identifying promising plant-based combinations (astragaloside IV, ginsenoside Rg1, and moringa leaf extract) that enhance exhaustive swimming time, grip strength, and muscle glycogen reserves. Findings on differential metabolites and short-chain fatty acids suggest plausible mechanisms, including reduced lactate, LDH, and CK levels alongside improved glycaemic homeostasis, mediated by gut microbiota and key metabolic pathways such as arginine-proline metabolism. These results align coherently with the pathophysiology of hypoxia in sports physiology.

Nevertheless, significant heterogeneity persists across the 49 included studies, arising from varied animal models, disparate doses, and mixed outcomes, which undermines indirect comparisons despite the use of SYRCLE for bias assessment. The high risk of bias in randomisation (10%) and incomplete data (47%) necessitates a more self-critical discussion of the reliability of SUCRA rankings. Extrapolation to humans at real altitude appears speculative, lacking data on oral bioavailability and genuine synergies of the mixtures; the selection of formulations, though logical, requires greater physiological explanation, please include these explanations.

The work offers practical value for functional interventions in hypoxia but demands a more cautious tone, emphasis on preclinical limitations, and clear delimitation of applications.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The researchers investigated whether a combination of plant-based bioactive compounds could improve athletic performance under hypoxic conditions and confirmed its effectiveness and mechanisms of action through experiments on mice.

Congratulations to the researchers on their extensive work.

Comments:

Dear authors,

1) It is imperative to ensure that the experimental phase of the study is articulated with greater clarity within the research objectives section of the introduction.

2) The experimental methodology described in lines 193-196 and 201-203 requires significant clarification:

Please correct the ascent velocity of "500 m/s" (line 194), as this is presumably a typographical error?
Opening the chamber for 2 hours daily (line 195) creates a model of intermittent hypoxia. The authors should justify this methodological choice and discuss how the daily normoxic window affects their findings.
Performing an exhaustive, weighted swimming test only 30 minutes post-gavage (lines 201-202) may induce severe acute stress or gastrointestinal issues, confounding the exercise performance data. The rationale for this brief interval needs to be explained.

3) Regarding the environmental parameters,

there is a significant discrepancy between the simulated altitude and the testing conditions. While the authors simulate a high-altitude environment of 5000 m (where ambient temperatures naturally drop well below 0°C), the exercise performance test was conducted in water maintained at 25°C (lines 202-203).
In real-world conditions at 5000 m, subjects would concurrently face extreme cold stress alongside hypobaric hypoxia. By maintaining a relatively warm water temperature of 25°C, the authors are isolating the effects of hypoxia but disregarding the synergistic physiological strain of thermal regulation. Please clarify if this choice was intentional to avoid the confounding variable of hypothermia, and discuss how this departure from realistic high-altitude conditions might limit the translatability of the findings.

4) Ln 387-388: A hypoxic mouse model was established by housing mice in a low-pressure chamber mimicking conditions at 5,500 m altitude. May I inquire whether the number of 5,500 m is accurate?

5) The employment of asterisks to signify statistical significance in Figures 6–9 is presently ambiguous. It is therefore requested that the specific significance levels (e.g. p-value thresholds) be defined in the corresponding figure captions in order to address this ambiguity.

In conclusion, while this study provides significant insights, the experimental framework requires more precise delimitation. It is essential to account for the multifaceted nature of high-altitude environments, where factors such as extreme thermal stress act synergistically with hypobaric hypoxia. Integrating these variables—beyond just oxygen deprivation—is necessary to ensure the ecological validity of the findings and their translatability to real-world conditions at 5000 meters.