Review Reports - Five-Day Supplementation with an Isotonic Beetroot Juice Drink Improves Sprint Interval Exercise and Muscle Oxygenation in Physically Active Individuals: A Randomized Crossover Trial

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study investigated the effects of isotonic beetroot juice supplementation over five days on sprint interval exercise performance and muscle oxygenation. The study is noteworthy as it provides a comprehensive evaluation that goes beyond the typical scope of sports nutrition research, assessing not only physical performance parameters such as sprint exercise capacity but also including a sensory evaluation of the isotonic beetroot juice used as a dietary supplement.

The study design, which builds upon prior research measuring salivary nitrate levels (as reported in Wong et al., 2024, Journal of Dietary Supplements), is particularly interesting in its inclusion of both plasma nitrate assessment and muscle oxygenation monitoring. This dual focus adds valuable insight into the mechanistic pathways of nitrate supplementation.

However, as the authors have acknowledged in the discussion section, the study could have been further strengthened by including measurements of pulmonary gas exchange. Assessing the correlation between muscle oxygenation and pulmonary gas exchange would have provided a more comprehensive understanding of the physiological responses to beetroot supplementation during high-intensity exercise.

In addition, while the ISO-C and ISO-BR formulations were appropriately matched for nitrate content and osmolality, it is somewhat unfortunate that they differed by up to 3 grams in carbohydrate content, which could potentially influence sprint performance outcomes. For future studies, it would be advisable to control for carbohydrate content when designing the control supplement formulation. Moreover, including a positive control such as arginine, which may also modulate nitric oxide production, would offer a valuable comparator to assess the specific efficacy of beetroot juice.

Overall, this study makes an important contribution to the field, and with consideration of these points in future research, it has the potential to further advance our understanding of nitrate supplementation in exercise performance.

Author Response

Responses to reviewers’ comments

We would like to thank the Reviewers for their careful review of our manuscript and their continued constructive feedback, which is very helpful. We have tried to respond thoroughly and to the best of our ability to the additional comments. We believe that the revision is significantly improved and hope it meets the reviewers' satisfaction. Below is our itemised response. C stands for Comment, R for Response. The page or line numbers provided are in reference to the revised manuscript version.

**Please refer to the attachment for the reply to the review report (Response to reviewers with reference) and manuscript revision.

Responses to Reviewer #1 (Highlighted in Green in the manuscript)

C1. However, as the authors have acknowledged in the discussion section, the study could have been further strengthened by including measurements of pulmonary gas exchange. Assessing the correlation between muscle oxygenation and pulmonary gas exchange would have provided a more comprehensive understanding of the physiological responses to beetroot supplementation during high-intensity exercise.

R1. We appreciate the reviewer's insightful comment regarding the inclusion of measurements of pulmonary gas exchange. We acknowledge that assessing the correlation between muscle oxygenation and pulmonary gas exchange would have provided a more comprehensive understanding of the physiological responses to beetroot supplementation during high-intensity exercise. However, we were unable to incorporate these measurements. We acknowledge and included this as a limitation. Future studies should consider including pulmonary gas exchange measurements to further elucidate the interplay between muscle oxygenation and pulmonary gas exchange during exercise. (Lines 589-594)

C2. In addition, while the ISO-C and ISO-BR formulations were appropriately matched for nitrate content and osmolality, it is somewhat unfortunate that they differed by up to 3 grams in carbohydrate content, which could potentially influence sprint performance outcomes. For future studies, it would be advisable to control carbohydrate content when designing the control supplement formulation.

R2. Thank you for your comment regarding the carbohydrate content in the ISO-C and ISO-BR formulations. While we ensured that these formulations were appropriately matched for nitrate content and osmolality, we recognized the difference in carbohydrate levels. The carbohydrates in ISO-BR primarily come from sugars, starches, and fiber, specifically derived from beetroot juice concentrate. We also compared the total sugar content and found that ISO-BR contains 1.8 grams less sugar than ISO-C. For future studies, we will incorporate the control for carbohydrate content when designing the control supplement formulation to minimize any potential confounding effects. (Table 1, Lines 292-298). We have added this as a possible limitation (Lines 618-621).

C3. Moreover, including a positive control such as arginine, which may also modulate nitric oxide production, would offer a valuable comparator to assess the specific efficacy of beetroot juice.

R3. Thanks for the insightful comment regarding the inclusion of a positive control such as arginine. We acknowledge that arginine would offer a valuable comparator to assess the specific efficacy of beetroot juice. However, due to the scope and design of our current study, we did not include arginine as a positive control. We agree that future studies can consider incorporating arginine or other relevant comparators to provide a more comprehensive evaluation of the effects of beetroot juice on nitric oxide production and exercise performance.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Please consider the following comments/observations/suggestions as an attempt to aid in the crafting of the manuscript. None of the following is meant to be judgmental or offered in a negative tone.

Overall, I found the manuscript to be sound and well organized. There are some aspects that I believe need address. Please see below:

Lines 11–25: Consider condensing the abstract slightly to emphasize key results and their implications more succinctly.

Ensure consistent use of abbreviations like ISOBR and ISOC after first mention (e.g., see Lines 24–25 and throughout Results/Discussion).

Line 63: “ingestion with higher nitrate doses on improving HIIT/SIT” consider revising to “ingestion of higher nitrate doses may improve HIIT/SIT performance.”

Lines 94–98: The primary and secondary aims should be stated more clearly. Consider explicitly stating: “The primary hypothesis was that ISOBR would improve power output and muscle oxygenation compared to ISOC.”

Lines 116–121: The power calculation is appreciated. However, the rationale for expecting a similar effect size in this study (based on a different protocol) could be elaborated for better justification.

Line 153: Consider rewriting: “The drink manufacturer coded both drinks, and the order of consumption was blinded to both researchers and participants.” for clarity.

Figure 1 (Line 171) and Figure 4 (Line 415): Add clearer titles and possibly legends summarizing key observations, which help standalone understanding.

Lines 421–436: The sensory findings are interesting but underexplained in terms of implications for real-world application. Suggest briefly stating whether palatability might affect long-term adherence.

Lines 476–504: The lack of plasma nitrite increase despite elevated nitrate and salivary NOx levels is noteworthy. This discrepancy deserves deeper exploration. Consider discussing whether methodological limitations in plasma sampling or conversion rates in tissues might explain this.

Lines 503–552: The discussion around the null findings in power output would benefit from deeper engagement with literature that found similar null effects, possibly categorizing differences in protocols, populations, or dosages.

Lines 556–570: The interpretation of the ∆HHb increase is not entirely clear. Was this an indicator of improved muscle oxygen extraction or a sign of higher oxygen demand? Clarify this point to strengthen the physiological relevance.

Lines 578–598: Well-identified limitations. It may be worth adding a statement about the small female sample (7 of 20), given sex-based physiological differences in nitrate metabolism.

Lines 600–609: These are well-stated. You might enhance the impact by noting any sports or situations (e.g., sprint cycling, football) where faster time to peak power could confer a competitive edge.

Author Response

Responses to reviewers’ comments

**Please refer to the attachment for the reply to the review report (Response to reviewers with reference) and manuscript revision.

Responses to Reviewer #2 (Highlighted in Red in the manuscript)

C1. Lines 11–25: Consider condensing the abstract slightly to emphasize key results and their implications more succinctly.

R1. We appreciate the reviewer's suggestion to condense the abstract. We have ensured that the abstract meets the word count requirements and highlights all important findings. The abstract has already been condensed to emphasize key results and their implications succinctly. (Lines 11-24, deleted line 22-23 in original manuscript).We believe that it is important to include the data for the primary finding of time to peak power rather than just inferential statistics.

C2. Ensure consistent use of abbreviations like ISOBR and ISOC after first mention (e.g., see Lines 24–25 and throughout Results/Discussion).

R2. We appreciate the reviewer's suggestion regarding the consistent use of abbreviations. Amendments have been made throughout the manuscript to ensure that all instances follow the abbreviations of isotonic drink (ISO-C) or isotonic beetroot juice drink (ISO-BR) after the first mention.

C3. Line 63: “ingestion with higher nitrate doses on improving HIIT/SIT” consider revising to “ingestion of higher nitrate doses may improve HIIT/SIT performance.”

R3. We agree with the reviewer's suggestion to revise the phrase “ingestion with higher nitrate doses on improving HIIT/SIT” to “ingestion of higher nitrate doses may improve HIIT/SIT performance.” Amendments have been made accordingly. (Lines 62-63)

C4. Lines 94–98: The primary and secondary aims should be stated more clearly. Consider explicitly stating: “The primary hypothesis was that ISOBR would improve power output and muscle oxygenation compared to ISOC.”

R4. We agree with the reviewer's suggestion to state the primary and secondary aims more clearly. Amendments have been made accordingly to explicitly state: “The primary hypothesis was that ISO-BR would improve power output and muscle oxygenation compared to ISO-C.” (Lines 107-109)

C5. Lines 116–121: The power calculation is appreciated. However, the rationale for expecting a similar effect size in this study (based on a different protocol) could be elaborated for better justification.

R5. We appreciate the reviewer's comment regarding the power calculation. The rationale for expecting a similar effect size in this study is based on the use of similar sports modalities, specifically sprint interval exercise, with power output as the outcome. Although there is variation in the number of sprints and sprinting time, previous research has demonstrated significant improvements in power output with beetroot juice supplementation under comparable conditions. Amendments have been made to elaborate on this justification. (Lines 135-138)

C6. Line 153: Consider rewriting: “The drink manufacturer coded both drinks, and the order of consumption was blinded to both researchers and participants.” for clarity.

R6. We appreciate the reviewer's recommendation to elaborate on the blinding process. The drink manufacturer labelled both beverages and assigned a unique code to each. The order of consumption was randomized, and this information remained concealed from both the researchers and participants to ensure unbiased results. This careful blinding procedure was meticulously implemented to minimize any potential bias in the study's outcomes. Amendments have been made for better clarity. (Lines 165-169)

C7. Figure 1 (Line 171) and Figure 4 (Line 415): Add clearer titles and possibly legends summarizing key observations, which help standalone understanding.

R7. We appreciate the reviewer's suggestion to provide clearer titles and legends. Amendments have been made to include titles and legends that summarize key observations more effectively. (Lines 187-191 and Lines 430-433)

C8. Lines 421–436: The sensory findings are interesting but underexplained in terms of implications for real-world application. Suggest briefly stating whether palatability might affect long-term adherence.

R8. Thank you for the suggestion. Amendments have been made to include the implications for real-world application: While our previous research demonstrated a significant preference for ISO-BR over beetroot juice, participants in this study expressed lower palatability for ISO-BR compared to ISO-C, an isotonic drink that is well-received by consumers in Singapore. Given that palatability may influence long-term adherence to ISO-BR, we recommended that the drink manufacturer explore sensory enhancements in future studies. (Lines 517-522)

C9. Lines 476–504: The lack of plasma nitrite increase despite elevated nitrate and salivary NOx levels is noteworthy. This discrepancy deserves deeper exploration. Consider discussing whether methodological limitations in plasma sampling or conversion rates in tissues might explain this.

R9. We thank the reviewer for highlighting this important point. While nitrite is a key intermediate in the nitrate–nitrite–NO pathway, its levels can be influenced by multiple factors, including rapid tissue uptake and conversion to nitric oxide, renal clearance, and potential methodological variability in sample handling and storage. In particular, plasma nitrite is known to be labile and sensitive to oxidative degradation, which could contribute to variability in measured concentrations. Additionally, nitrite may be rapidly utilized in peripheral tissues where it exerts biological effects, thereby reducing its accumulation in circulation (Tsikas, 2012). We have now added a discussion of these potential explanations to the revised manuscript (Lines 504-510)

C10. Lines 503–552: The discussion around the null findings in power output would benefit from deeper engagement with literature that found similar null effects, possibly categorizing differences in protocols, populations, or dosages.

R10. We appreciate the reviewer's comment regarding the discussion around the null findings in power output. Literature with similar null effects has been included, and we have added a comment about variations in protocols, populations, and dosages may restrict a firm conclusion of evidence for the effect of beetroot on power output during repeated sprints. (Lines 530-535)

C11. Lines 556–570: The interpretation of the ∆HHb increase is not entirely clear. Was this an indicator of improved muscle oxygen extraction or a sign of higher oxygen demand? Clarify this point to strengthen the physiological relevance.

R11. Thank you for the comment. The physiological explanation was included for clarity in the manuscript. It is hypothesized that ISO-BR may accelerate ATP (Adenosine triphosphate)/PCr (Phosphocreatine) turnover and enhance PCr recovery between repetitions. This rapid PCr breakdown suggests increased oxygen extraction, potentially coupled with improved oxygen delivery, thereby optimizing mitochondrial respiration efficiency for ATP regeneration during SIE and promoting a shift towards anaerobic ATP resynthesis. (Lines 573-577)

However, we emphasize that this is a hypothesis and recognize that we did not directly measure ATP/PCr turnover or recovery in this experiment.

C12. Lines 578–598: Well-identified limitations. It may be worth adding a statement about the small female sample (7 of 20), given sex-based physiological differences in nitrate metabolism.

R12. Thanks for the suggestion. We have included a statement regarding the small female sample in this study. The small number of female subjects (7 out of 20) in the current study makes comparisons within this sex uncertain, while comparisons between sexes would be severely underpowered and prone to Type 2 error. However, other prior research has indicated that the salivary and plasma responses of nitrate and nitrite following the ingestion of dietary nitrate are consistent across both sexes (Bondonno et al., 2015). Nevertheless, a substantial body of research comparing the impact of nitrate on different sexes has not been undertaken, and future studies should investigate this issue in more detail. (Lines 611-618)

C13. Lines 600–609: These are well-stated. You might enhance the impact by noting any sports or situations (e.g., sprint cycling, football) where faster time to peak power could confer a competitive edge.

R13. Thank you for the suggestion. We have included your suggestion in the manuscript. (Line 634)

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors are reporting on a double blinded cross over study on the effect of 5 days of Beetroot Nitrate supplementation on repeated cycling sprint interval exercise. The manuscript is strongly justified and well positioned within the relevant literature on nitrate supplementation. The methodology as reported by the authors is clearly replicable and potential biases where possible have sought to be controlled. The writing is clear and the arguments are well presented.

Ln 169; The text in Figure 1 is not legible, either increase the size of font used and or increase the size of the figure.

Ln 354; At this point in the results the authors need to define as to why some measures are an n=18 (Figure 3 and Table 3) while others are n=20.

Ln 395, Figure 3; The clarity of the figure needs to be substantially improved. X and Y axis labels and titles need to be larger so that they are readable at the size of the inserted figure. Suggest using only solid black and open white columns.

Ln 415, Figure 4; The clarity of the figure needs to be substantially improved. X and Y axis labels and titles need to be larger so that they are readable at the size of the inserted figure. For the data presented in these panels, a box and whiskers plot would be more informative to the reader.

Ln 427, Table 2; Considering the drink preference outcome should this have been applied as a covariate in the analysis?

Ln 451, Figure 5; The clarity of the figure needs to be substantially improved as per suggestions provided for figures 1, 3 and 4. How is the data as presented different between what is shown in this figure and the data displayed in Table 3?

Author Response

Responses to reviewers’ comments

**Please refer to the attachment for the reply to the review report (Response to reviewers with reference) and manuscript revision.

Responses to Reviewer #3 (Highlighted in Blue in the manuscript)

C1. Ln 169; The text in Figure 1 is not legible, either increase the size of font used and or increase the size of the figure.

R1. Thank you for the feedback. Our apologies for this. The font size has been increased and bolded to enhance readability. (Figure 1, Lines 184-185)

C2. Ln 354; At this point in the results the authors need to define as to why some measures are an n=18 (Figure 3 and Table 3) while others are n=20.

R2. Thank you for the feedback and agreed. All participants completed the SIE test. However, due to connection interruptions and unsuccessful blood draws, muscle oxygenation data and blood plasma data were missing for two instances. Consequently, only 18 participants were included in the analysis of muscle oxygenation and plasma nitrate/nitrite levels. We have included this statement in the manuscript. (Lines 366-373)

C3. Ln 395, Figure 3; The clarity of the figure needs to be substantially improved. X and Y axis labels and titles need to be larger so that they are readable at the size of the inserted figure. Suggest using only solid black and open white columns.

R3. Thank you for your feedback on Figure 3. We appreciate your suggestions for improving the clarity of the figure. We have made the following adjustments: a) Increase the size of the X and Y axis labels and titles to ensure they are readable. b) Use only solid black and open white columns for better visual distinction. We hope these changes enhance the readability and overall quality of the figure. (Figure 3, Lines 406-407)

C4. Ln 415, Figure 4; The clarity of the figure needs to be substantially improved. X and Y axis labels and titles need to be larger so that they are readable at the size of the inserted figure. For the data presented in these panels, a box and whiskers plot would be more informative to the reader.

R4. Thank you for your feedback on Figure 4. We appreciate your suggestions for improving the clarity of the figure. We have made the following adjustments: a) Increase the size of the X and Y axis labels and titles to ensure they are readable. b) Use only solid black and open white columns for better visual distinction. We hope these changes enhanced the readability and overall quality of the figure. As our primary outcomes are the comparison of mean power outputs, we suggest that using a bar chart for means comparison is adequate. (Figure 4, Line 427-428)

C5. Ln 427, Table 2; Considering the drink preference outcome should this have been applied as a covariate in the analysis?

R5. Thank you for your insightful comment regarding the inclusion of drink preference as a covariate in the analysis. This study was designed as a double-blinded trial, in which participants nor researchers were aware of the specific test drinks. This design was implemented to minimize bias and enhance the reliability of our results. We conducted an exit survey to further evaluate the integrity of the blinding. Participants were asked to guess which drink they have received and its expected effect. The results of the survey indicated that 50% of the participants failed to correctly guess the drink type and its efficacy, indicating effective blinding. Given the rigor of blinding procedures and evidence from the exit survey, we do not believe that drink preference meaningfully confounded the results. Therefore, we consider that adjusting for drink preference as a covariate in the analysis is not necessary. (Line 367-373)

C6. Ln 451, Figure 5; The clarity of the figure needs to be substantially improved as per suggestions provided for figures 1, 3 and 4. How is the data as presented different between what is shown in this figure and the data displayed in Table 3?

R6. Thank you for your feedback on Figure 5. We appreciate your suggestions for improving the clarity of the figure. We have made the following adjustments: a) Increase the size of the X and Y axis labels and titles to ensure they are readable. b) Use only solid black and open white columns for better visual distinction. We hope these changes enhanced the readability and overall quality of the figure. Figure 5 and Table 3 presented different outcomes. Figure 3 displays the relative changes of ΔTSI, ΔO2Hb, ΔHHb, and ΔtHb which were tabulated by subtracting the value at specified time points (last 5 seconds of each stage) from baseline during the resting period before performing the exercise protocol. Conversely, Table 3 shows the magnitude of ΔTSI, ΔO2Hb, ΔHHb and ΔtHb which were calculated based on the difference between the baseline of each stage (5 seconds before the start of each stage) and the endpoint (5 seconds before the end of each stage) (the difference between the baseline and the endpoint of each stage was interpreted as the rate of change of each stage). The magnitude analysis aims to understand the specific changes (i.e., the magnitude or rate of change) in each stage of exercise and recovery. (Line 267-278)

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

The submitted work examines how recreationally active people's performance metrics and muscle oxygenation are affected after five days of supplementing with an isotonic beverage made with beet juice, which is high in nitrates. The paper is based on a double-blind, randomized crossover trial that was conducted with meticulous methodological attention to detail and has well-presented results.

However, there are some issues I would like you to address:

It is challenging to draw firm conclusions on the function of the medium itself (isotonicity vs. nitrate content) because a third control group is absent (e.g., placebo water or a drink with nitrates but non-isotonic). Please go into great depth about the limitations of this group's absence and how it affects how ISO-BR impacts are interpreted.
It's intriguing that an isotonic beverage can be used as a vehicle for nitrate supplementation, but the introduction does not adequately highlight the study's novelty.
Bring up the possible impact of the workout regimen's duration on the effectiveness of nitrate supplementation, as a 60-second muscular training period might not result in a change in maximal power. Your explanation for the study protocol selection would be greatly appreciated.
I propose broadening the conclusions to encompass real-world applications, like sports, where a shorter time to peak power might be significant, or including a comment in the discussion regarding the need to optimize further ISO0BR's taste, which was less acceptable.
I support improving the graphical representations of results to make them appear more appealing and facilitate the reader's comprehension of the content.

Best regards,

The reviewer.

Author Response

Responses to reviewers’ comments

**Please refer to the attachment for the reply to the review report (Response to reviewers with reference) and manuscript revision.

Responses to Reviewer #4 (Highlighted in Purple in the manuscript)

C1. It is challenging to draw firm conclusions on the function of the medium itself (isotonicity vs. nitrate content) because a third control group is absent (e.g., placebo water or a drink with nitrates but non-isotonic). Please go into great depth about the limitations of this group's absence and how it affects how ISO-BR impacts are interpreted.

R1. Thank you for your comment. We acknowledged that the study designs were restricted to comparing ISO-BR with ISO-C, excluding the evaluation of ISO-BR against an isotonic low nitrate placebo or water as a control in the study design. However, this was a deliberate decision informed by prior evidence. Our earlier study investigated the effect of isotonicity by comparing an isotonic beetroot juice drink (ISO-BR) with a similar non-isotonic beetroot juice drink (BR) to determine whether isotonicity enhances nitrate-nitrite bioavailability and high-intensity exercise performance.

The present study was designed to examine whether the inclusion of beetroot juice in an isotonic sports drink (referred to as ISO-C) is beneficial to performance compared with an isotonic drink itself. A low nitrate placebo control was not included as findings from several systematic reviews, including our own (Wong et al., 2021), and studies by others have shown a consistent improvement with nitrate (beetroot juice) on several aspects of exercise performance (Bailey et al., 2009; Cermak et al., 2012; Cuenca et al., 2018; Dominguez et al., 2017; Jodra et al., 2020; Lansley et al., 2011; Nyakayiru et al., 2017). These include time to exhaustion, endurance exercise performance, distance covered in Yo-Yo IR1, and peak power output of isolated single sprints after BR supplementation.

While a more comprehensive 2×2 factorial crossover design (manipulating both nitrate content and isotonicity) would allow for deeper mechanistic exploration, the objective of this study was to isolate the effect of high-nitrate beetroot formulation in isotonic drink. Accordingly, the sample size was powered to detect differences between two drinks rather than four experimental conditions. Taken together, our previous study (Wong et al., 2024) manipulated isotonicity in beetroot juice, and this study evaluated the impact of nitrate content within an isotonic sports drink framework. These complementary designs together provide novel insights and lay the groundwork for future research to investigate underlying physiological mechanisms. We have acknowledged this design limitation and the need for more mechanistic work in the discussion section of the manuscript (Lines 596–606).

C2. It's intriguing that an isotonic beverage can be used as a vehicle for nitrate supplementation, but the introduction does not adequately highlight the study's novelty.

R2. The primary goal of this study was to evaluate the ergogenic impact of dietary nitrate from a novel isotonic beetroot juice drink on high-intensity exercise performance. Most studies examining the effect of nitrate on exercise performance have utilized beetroot juice as the primary source of nitrate. The provision of nitrate in a beetroot juice drink that was isotonic may be beneficial in terms of increased salivary flow rate and oral microbiome activity, and in relation to gastric emptying, which may enhance nitrate/nitrite reduction in the gut. This might provide performance advantages over beetroot juice alone. We have included the paragraph below in the introduction section.

“Approximately 25% of ingested dietary nitrate is absorbed by the salivary glands and reduced to nitrite by oral bacteria, which can be converted to nitric oxide (NO) in the stomach or enter systemic circulation (Jones et al., 2021). The use of antibacterial mouthwash has been shown to reduce plasma nitrite concentrations post-ingestion, likely by disrupting oral microbiota (Blot, 2021). Thus, enhancing oral microbiome activity could promote NO production. Delivering nitrate through an isotonic drink may be beneficial, as these drinks improve gastric emptying and nutrient absorption (Leiper, 2015). Nitrate combined with ascorbic acid in an isotonic drink may also enhance NO production in the stomach (Peri et al., 2005). Additionally, isotonic drinks can increase salivary flow rates (Bishop et al., 2000; Siow et al., 2017), promoting the transfer of nitrite into circulation.” (Lines 65-74)

C3. Bring up the possible impact of the workout regimen's duration on the effectiveness of nitrate supplementation, as a 60-second muscular training period might not result in a change in maximal power. Your explanation for the study protocol selection would be greatly appreciated.

R3. In our prior systematic review and meta-analysis, the impact of dietary nitrate from beetroot juice on high-intensity interval training (HIIT) and sprint interval training (SIT) was assessed. The results indicated no evidence for acute or chronic beetroot juice intake to enhance performance, as measured by power output, during these high-intensity exercises (Wong et al., 2021). Variations in the exercise protocols, nitrate dosage, type of beetroot products, supplementation strategy, and duration restrict a firm conclusion of evidence for an effect of beetroot on power output during repeated sprints at present. However, interestingly, a subgroup analysis found that a higher total nitrate dose from chronic beetroot supplementation was correlated with a greater increase in circulating plasma nitrite. This suggests potential nitrate and nitrite storage within the body as NO reservoirs with increased daily intake (Wong et al., 2021). In addition, our prior study found that consuming beetroot in the form of an isotonic drink enhanced peak power output during isolated cycling sprints by 3.9% compared to beetroot juice alone (Wong et al., 2024). Another study we conducted found that ISO-BR improved high-intensity cycling time-trial performance compared to ISO-C in the 5- to 30-minute regime (currently under review). The potential ergogenic effects of isotonic beetroot juice drinks warrant further exploration, particularly for repeated high-intensity sprints using chronic (multi-day) supplementation. In addition, research has shown that shorter exercise work bouts (5-15 seconds) targeting peak power generation can promote significant increases in energy expenditure (Islam et al., 2018) and promote significant acute cardiorespiratory responses (Danek et al., 2020) compared to 30-second SIE protocols. Conversely, short exercise work bouts with short recovery periods (30 to 60 seconds) not only simulate higher acute cardiorespiratory responses (Danek et al., 2020) but are also able to maintain the same performance in the sprint interval protocol (Olek et al., 2018; Ratel et al., 2002). In addition, short exercise work bouts (5-15 seconds) targeting peak power generation have been used in several previous beetroot juice/nitrate research studies (Aucouturier et al., 2015; Bernardi et al., 2018; Kokkinoplitis & Chester, 2014; Martin et al., 2014; Muggeridge et al., 2013; Smith et al., 2019; Wylie et al., 2016). As the primary aim of the study is to evaluate the effects of ISO-BR and ISO-C on SIE, particularly on power output, a 60-second SIE was deemed suitable. We have briefly communicated some of this information in the manuscript. (Lines 248-252)

C4. I propose broadening the conclusions to encompass real-world applications, like sports, where a shorter time to peak power might be significant.

R4. Thanks for the suggestion. We have included the example of sports as suggested by Reviewer 2 (please refer to R13 under Reviewer 2) and line 634 in the manuscript.

C5. Including a comment in the discussion regarding the need to optimize further ISO-BR's taste, which was less acceptable.

R5. Thank you for your suggestion. Considering that palatability may influence long-term adherence to ISO-BR, we recommended that the drink manufacturer to explore sensory enhancements in future studies. We have included this suggestion to further optimize the taste in future research. (Line 517-522).

C6. I support improving the graphical representations of results to make them appear more appealing and facilitate the reader's comprehension of the content.

R6. Thank you for your feedback. We appreciate your suggestions for improving the clarity of the figure. We have made the adjustments according to the recommendations by reviewer 3 (R1, R3, R4 & R6) to improve our graphics. We hope these changes enhanced the readability and overall quality of the figure.

Author Response File: Author Response.pdf

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for your responses.

Best regards,

The reviewer.