Review Reports - Resetting Time: The Role of Exercise Timing in Circadian Reprogramming for Metabolic Health

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review article describes a comprehensive overview of the skeletal muscle circadian clock and emphasizes the importance of timely exercise in maintaining circadian metabolic health. This review article contains four crucial areas, including skeletal muscle circadian clock, consequences of circadian misalignment, importance of timely exercise, and the relevant use of omics tools in muscle circadian clock research. The introduction provides an explicit hierarchical nature of the circadian clock and a concise outline of the article, citing both classic and recent references. Figure 1 clearly illustrates the central aim of the article – how timed exercise influences circadian metabolic health. In the section on skeletal muscle clock, the author describes the molecular nature of the circadian clock and metabolic consequences observed in skeletal muscle-specific Bmal1 knockout mice, and clock alterations in metabolic disorders such as obesity and T2DM. The section also describes the interaction between clock components and metabolic pathways, emphasizing skeletal muscle as a central regulator that can influence circadian rhythms in other tissues, acting as a temporal coordination hub within the body. The second section, "consequences of circadian misalignment due to behaviour, genetic defects and nutrient overload," describes how clock disruption contributes to metabolic diseases, such as obesity and T2DM. All the cited references are relevant and up to date. The third section is the primary interest of the article. While much of the literature has focused on time-restricted feeding (TRF) as a simple and effective intervention for metabolic disorders, this article highlights timely exercise as an equally powerful tool for improving circadian alignment. Figure 2 clearly supports the aim of the article, showing how exercise influences molecular pathways linked to circadian function. One of the promising studies in the section is Chaix et al., who used combined timed exercise and food can correct circadian misalignment and improve metabolic flexibility. The authors suggest that future research will increasingly focus on integrating these chronotherapeutic tools for the prevention and treatment of circadian-related metabolic diseases. The final section discusses the application of omics techniques in circadian muscle research and obesity, providing extensive references that will benefit readers interested in advanced methodologies.

Overall, this review is timely and relevant, especially given the growing interest in timed exercise as a therapeutic strategy for circadian and metabolic disorders. It thoroughly covers most of the key literature and offers a clear, well-structured summary of the field. The manuscript appears ready for publication after addressing a few minor points.

Minor Comments.

Some sentences require proper references for clarity, because it is unclear whether they reflect the author's interpretation or previously published findings. For example:
- Page 9, lines 342 and 350
- Page 10, line 370
- Page 12, line 459
- Page 12, line 478
One important area missing from the review is muscle loss (sarcopenia) and its circadian regulation. Incorporating literature on sarcopenia and its metabolic consequences would make the article a more comprehensive resource on the skeletal muscle circadian clock.

Author Response

Thank you for your thoughtful and constructive review of the manuscript. I greatly appreciate your recognition of the review’s scope, structure, and relevance, particularly your comments on the clarity of the introduction, the strength of the figures, and the integration of recent literature. I am especially pleased that you found the emphasis on timed exercise as a chronotherapeutic strategy both timely and valuable.

Your encouraging feedback on the manuscript’s readiness for publication is truly appreciated. I have carefully addressed the minor points raised and made the necessary revisions to ensure clarity, proper referencing, and improved precision where needed. A detailed response to each point follows below in bold red text.

Minor Comments.

Some sentences require proper references for clarity, because it is unclear whether they reflect the author's interpretation or previously published findings. For example:

Page 9, lines 342 and 350
Page 10, line 370
Page 12, line 459
Page 12, line 478

Thank you for highlighting these instances. Each of the indicated sentences have carefully been reviewed and now have a clarified source by adding appropriate references where applicable. There is also a slight revision of language in the interest of clarity. It is hoped these changes help improve transparency and ensure that all claims are properly supported. Below, is a line-by-line response:

Line 342 – Reference included, Hitrec et al (2023). This study demonstrates timed daily voluntary wheel running in rodents synchronised circadian rhythms, producing more stable and robust 24‑hour activity patterns in mice. This illustrates that exercise not only shifts phase but consolidates and amplifies behavioural circadian amplitude, reflecting enhanced circadian robustness.

Line 350 – Updated text in red and provided appropriate reference (Lamia et al 2010). This study demonstrated that activating AMPK reduced nuclear CRY1, decreased CRY1–PER2 binding, increased CRY1 ubiquitination by FBXL3, and altered circadian rhythm parameters both in vivo and in vitro.

Line 370 – This sentence is in summary of previously discussed study. Have added the reference to the line for clarity.

Line 459 – Cited Zhang et al (2014), which provides experimental evidence supporting the use of integrated transcriptomic, proteomic, and metabolomic approaches to uncover circadian regulation in metabolic pathways.

Line 478 - Cited Sato et al, (2019). This study shows that exercise timing affects transcriptional and metabolic pathways in skeletal muscle differently depending on time of day, suggesting the need for personalised, time-based interventions.

One important area missing from the review is muscle loss (sarcopenia) and its circadian regulation. Incorporating literature on sarcopenia and its metabolic consequences would make the article a more comprehensive resource on the skeletal muscle circadian clock.

Thank you for this valuable suggestion. In response, incorporated is a brief discussion on the circadian regulation of sarcopenia into the section outlining the role of skeletal muscle in metabolic health (line 183, in red text). Specifically, it is noted that circadian disruption has been implicated in age-related muscle loss, with evidence linking diminished clock gene expression to anabolic resistance and mitochondrial dysfunction. This addition acknowledges the metabolic consequences of sarcopenia and aligns with the review’s broader focus on the role of muscle clocks in systemic energy regulation. A supporting reference (Silva et al., 2021) has also been included to ground this discussion.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a well-written manuscrip. The author provided up-to-date and in-depth discussions on research on circadian rhythm in the muscle and the impact of exercise on muscle physiology and organismal health. The potential application of personalized exercise scheduling to improve health (e.g., to ameliorate metabolic dysfunctions) is also an important suggestion. The overall quality of the manuscript is high. I have only a few minor suggestions.

Myokines (i.e., Il6) are only briefly mentioned in the manuscript. The authors could elaborate more on irisin and its benefical effects to health.
Line 287-289: In mice, both acute and chronic bouts 287
of exercise during the early active phase elicit greater improvements in exercise capacity, substrate utilisatsion, and gene expression than equivalent training performed later in the day. This statement appears inaccurate. Please check reference [17] for the details.
Line 348-350: AMPK activation in response to energetic stress enhances nuclear translocation of CRY1 and promotes PER2 degradation, thereby modulating the pace of the molecular clock. This statement appears inaccurate. Please check reference [56] for the details.

Author Response

Thank you very much for your positive and encouraging assessment of the manuscript. I appreciate your careful reading and insightful suggestions, which have helped improve both the clarity and accuracy of the manuscript. Please find responses to each of your specific points below in bolded red text.

Myokines (i.e., Il6) are only briefly mentioned in the manuscript. The authors could elaborate more on irisin and its benefical effects to health.

Thank you for this suggestion. A brief paragraph discussing irisin has now been added to the relevant section (line 232, following discussion of myokines). The revision highlights the potential circadian influence of irisin, its role in mediating exercise-induced metabolic benefits, and its potential relevance to systemic energy homeostasis and neuroprotection. An appropriate reference has been included to support this addition (Bostrom et al, 2012).

Line 287-289: In mice, both acute and chronic bouts 287
of exercise during the early active phase elicit greater improvements in exercise capacity, substrate utilisatsion, and gene expression than equivalent training performed later in the day. This statement appears inaccurate. Please check reference [17] for the details.

Thank you for pointing this out. Upon re-reviewing reference [17] (Adamovich et al.), you are correct that the late active phase, rather than the early phase, yielded superior outcomes in terms of exercise performance and metabolic gene expression in that study. The sentence has now been corrected to accurately reflect these findings:

‘In mice, both acute and chronic bouts of exercise during the late active phase elicit greater improvements in exercise capacity, substrate utilisation, and gene expression than equivalent training performed earlier in the day [17].’

Line 348-350: AMPK activation in response to energetic stress enhances nuclear translocation of CRY1 and promotes PER2 degradation, thereby modulating the pace of the molecular clock. This statement appears inaccurate. Please check reference [56] for the details.

Thank you for flagging this. You are correct that reference [56] (Jordan & Lamia, 2013) and earlier work (Lamia et al, 2009) show that AMPK phosphorylates CRY1, leading to its destabilisation and degradation, not nuclear import. The sentence has been revised and highlighted in red in the manuscript for accuracy.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This is a beautifully written review article that integrates basic biomedical research on biological clocks with translational applications for treating metabolic disease using novel chronotherapeutic approaches. The author provides an excellent summary of circadian biological clock mechanisms, including the central light-entrainable pacemaker, peripheral oscillators, and the concept of a circadian system with coupling mechanisms among constituent oscillators with an emphasis on muscle circadian clocks, followed by an insightful integration of basic circadian biology with obesity, metabolic disorder, exercise with respect to the role of circadian synchrony- and then goes on to propose a personalized omics-based chronotherapeutic approach to applying these principles in clinical practice. Human and non-human research is well integrated, and the logic of the review also suggests directions for further research. The potential for reversibility of muscle clock-driven circadian desynchrony (line 259) is an especially important point, as is the potential for personalized chronotherapy (lines 410-427).

The only suggestions I have for the author's and editor's consideration are:

1) a suggestion to expand on the concept of a "holistic strategy to correct misaligned rhythms", involving "optimally timed physical activity, feeding-fasting cycles, and sleep hygiene" (lines 424-427) to include some additional discussion to note that further research on non-photic zeitgebers is needed to develop optimal protocols that maximize therapeutic effects and minimize possible harmful side-effects of different time-restricted feeding and fasting cycles, exercise regimes and photic and pharmacological methods of optimizing sleep hygiene. Some time-restricted feeding regimes may be harmful, too much exercise at the wrong times may disrupt circadian synchrony, and pharmacological and photic treatments intended to enhance sleep hygiene may also disrupt circadian synchrony or be contraindicated by certain psychiatric disorders if they are misapplied, for example. In other words, anything that is capable of doing good is capable of doing harm, so the idea of employing chronotherapeutic approaches also has inherent risks and potential side-effects, and in this context, exercise, diet regimes and possible photic treatments (for instance with regard to sleep hygiene) should be treated, effectively, as if they were drugs- and their misapplication, or interactions among them may be harmful.

2) The author might consider, in the Discussion, more explicit guidance on the most useful questions for further basic and clinical research to support further progress along the lines proposed for chronotherapeutic approaches to metabolic disease.

Author Response

Thank you very much for your generous and thoughtful evaluation of the manuscript. I greatly appreciate your recognition of the integrative approach, clarity of logic, and translational emphasis. Your comments on the importance of circadian desynchrony reversibility and the potential of personalised chronotherapy are particularly encouraging. I am also grateful for your insightful suggestions, which have helped strengthen the manuscript. Below are detailed responses to each point in bolded red text.

The only suggestions I have for the author's and editor's consideration are:

a suggestion to expand on the concept of a "holistic strategy to correct misaligned rhythms", involving "optimally timed physical activity, feeding-fasting cycles, and sleep hygiene" (lines 424-427) to include some additional discussion to note that further research on non-photic zeitgebers is needed to develop optimal protocols that maximize therapeutic effects and minimize possible harmful side-effects of different time-restricted feeding and fasting cycles, exercise regimes and photic and pharmacological methods of optimizing sleep hygiene. Some time-restricted feeding regimes may be harmful, too much exercise at the wrong times may disrupt circadian synchrony, and pharmacological and photic treatments intended to enhance sleep hygiene may also disrupt circadian synchrony or be contraindicated by certain psychiatric disorders if they are misapplied, for example. In other words, anything that is capable of doing good is capable of doing harm, so the idea of employing chronotherapeutic approaches also has inherent risks and potential side-effects, and in this context, exercise, diet regimes and possible photic treatments (for instance with regard to sleep hygiene) should be treated, effectively, as if they were drugs- and their misapplication, or interactions among them may be harmful.

Thank you for this important and nuanced suggestion. I have revised the final paragraph of that section (lines 455–464, in red) to incorporate a cautionary note regarding the potential risks, side effects, and contraindications of chronotherapeutic interventions. The revised text now emphasises the need for additional research to determine optimal combinations, timing, and dosage of non-photic interventions, including exercise, feeding-fasting cycles, and light or pharmacological therapies, while also highlighting the importance of treating these tools with the same caution as pharmacological agents. A brief acknowledgment of individual variability and clinical context has also been added to underscore the potential for both benefit and harm depending on application. This aligns with your point that ‘anything capable of doing good is also capable of doing harm.’ Thank you again for this valuable addition.

The author might consider, in the Discussion, more explicit guidance on the most useful questions for further basic and clinical research to support further progress along the lines proposed for chronotherapeutic approaches to metabolic disease.

Thank you for this excellent recommendation. In response, I have added a brief concluding paragraph to the end of the discussion (beginning line 528, in red) highlighting several key directions for future research. These include: (1) determining optimal exercise timing across diverse chronotypes and metabolic states; (2) identifying biomarkers of circadian alignment and responsiveness to chrono-exercise; (3) characterising interactions between feeding, activity, and sleep timing at the molecular level; and (4) conducting long-term clinical trials of personalised, multi-zeitgeber interventions. These points are intended to provide a more explicit roadmap for advancing both basic circadian science and translational applications in metabolic disease.

Author Response File: Author Response.pdf