{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T05:11:25Z","timestamp":1772169085053,"version":"3.50.1"},"update-to":[{"DOI":"10.12688\/wellcomeopenres.23269.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,3,17]],"date-time":"2025-03-17T00:00:00Z","timestamp":1742169600000}}],"reference-count":48,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2025,3,17]],"date-time":"2025-03-17T00:00:00Z","timestamp":1742169600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["101550"],"award-info":[{"award-number":["101550"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["wellcomeopenresearch.org"],"crossmark-restriction":false},"short-container-title":["Wellcome Open Res"],"abstract":"<ns7:p>Background Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by mutations in the dystrophin gene resulting in cycles of muscle degeneration, inflammation and regeneration. The 6-minute walk test (6MWT) is a key functional outcome measure for DMD patient clinical trials and has been adapted for use in animal models of the disease. The DE50-MD dog model of DMD closely reflects the DMD patient phenotype prior to loss of ambulation. For pre-clinical trials using this model, functional outcome measures must be established. Methods This longitudinal study compared distance walked in a 6MWT by DE50-MD and WT control dogs and assessed the utility of the 6MWT as a functional biomarker. Dogs underwent two 6MWTs conducted approximately 48-hours apart, at 3, 6, 9, 12, 15 and 18 months of age. In addition, we evaluated the stability of selected blood-borne biomarkers in 12-month old DE50-MD and WT dogs 0, 3, 6, 24 and 48 hours following a 6MWT. Results DE50-MD dogs exhibited significantly shorter 6-minute walk distance (6MWD) than WT dogs at all timepoints (P&lt;0.05), with no difference in 6MWD between the first and second 6MWT. C-C motif chemokine ligand 2 (CCL2), myomesin-3 (MYOM3) and myostatin (MSTN) were biomarkers of the DE50-MD phenotype that remained unchanged in DE50-MD dogs following the 6MWT, while creatine kinase (CK) activity significantly increased 3-hours following the test in DE50-MD dogs but remained unchanged in WT dogs. Conclusions The 6MWT effectively discriminates DE50-MD from WT dogs aged 3-18 months and a single 6MWT is sufficient for future studies. Serum MYOM3, CCL2 and MSTN are good biomarkers of the DE50-MD phenotype that are unaffected by the relatively low level exertion performed in the 6MWT by 12-month-old DE50-MD dogs.<\/ns7:p>","DOI":"10.12688\/wellcomeopenres.23269.2","type":"journal-article","created":{"date-parts":[[2025,3,17]],"date-time":"2025-03-17T12:55:08Z","timestamp":1742216108000},"page":"681","update-policy":"https:\/\/doi.org\/10.12688\/wellcomeopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Evaluation of a six-minute walk test in the DE50-MD canine model of Duchenne muscular dystrophy and its effect on blood-borne biomarkers"],"prefix":"10.12688","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1614-504X","authenticated-orcid":false,"given":"Dominique","family":"Riddell","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rachel","family":"Harron","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2283-2118","authenticated-orcid":false,"given":"John","family":"Hildyard","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1425-6344","authenticated-orcid":false,"given":"Dominic","family":"Wells","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4344-6438","authenticated-orcid":false,"given":"Richard","family":"Piercy","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"2560","published-online":{"date-parts":[[2025,3,17]]},"reference":[{"key":"ref-1","doi-asserted-by":"publisher","first-page":"177-89","DOI":"10.1016\/S1474-4422(09)70272-8","article-title":"Diagnosis 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Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/9-681\/v2\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/9-681\/v2\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/9-681\/v2\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,7,26]],"date-time":"2025-07-26T02:25:08Z","timestamp":1753496708000},"score":1,"resource":{"primary":{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/9-681\/v2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,3,17]]},"references-count":48,"URL":"https:\/\/doi.org\/10.12688\/wellcomeopenres.23269.2","relation":{"has-version":[{"id-type":"doi","id":"10.12688\/wellcomeopenres.23269.1","asserted-by":"subject"}],"has-review":[{"id-type":"doi","id":"10.21956\/wellcomeopenres.26410.r121945","asserted-by":"subject"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.26410.r121945","asserted-by":"object"}]},"ISSN":["2398-502X"],"issn-type":[{"value":"2398-502X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,3,17]]},"assertion":[{"value":"Indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/9-681\/v2#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/wellcomeopenres.25648.r114903, Kristy Swiderski, The University of Melbourne, Parkville, VIC, 30 Dec 2024, version 1, 1 approved, 1 approved with reservations","URL":"https:\/\/wellcomeopenresearch.org\/articles\/9-681\/v1#referee-response-114903","order":0,"name":"referee-response-114903","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Dominique Riddell<\/b>; \n<i>Posted: 07 Mar 2025<\/i>; Thank you very much for your supportive feedback. We are happy to address your questions.  \n<b>1.&nbsp;<\/b>\n<i>As a minor point, analysis of serum biomarkers to determine the impact of the 6MWT was only performed in the 12-month-old dogs, which have undertaken 4 bouts of the 6MWT. Is it likely that the repeated testing may influence these markers? <\/i>  \n<b>Response:&nbsp;<\/b>Regarding the impact of 4 bouts of 6MWTs on the serum biomarkers: the distance walked by the dogs in the 6MWT is relatively short, with a percentage of the test spent resting, and the test is conducted infrequently, at 3-monthly intervals in animals that between tests have access to outside runs and grass paddocks when they can exercise freely. Thus, we deem it unlikely that the 6 minutes of mild, intermittent exercise would have an effect that lasts until the following test conducted 3-months later. However, it is possible that having performed the test 4 times, the dogs may remember previous tests and therefore better understand what is expected of them. In WT dogs, there is a reduced time spent not walking as dogs age, which we have speculated is due to learning what the test involves and\/or increased focus as the dogs mature. This effect is not seen in the DE50-MD dogs however, who actually spend more time not walking during the 6MWT as they age \u2013 likely due to reduced functional capacity of their muscle. Thus, we think it unlikely that repeated bouts of testing will influence analysis of serum biomarkers in 12-month-old DE50-MD dogs.  \n<b>2.&nbsp;<\/b>\n<i>Additionally, given the authors have previously assessed at least some of these biomarkers over these specified ages and demonstrated that some are not increased in younger dogs, is it possible that the 6MWT may have greater influence on these markers if tested in younger dogs? Acknowledgement of these points in the discussion would be useful.<\/i>  \n<b>Response:&nbsp;<\/b> Of the biomarkers measured in the study, we have previously shown that serum CCL2, MYOM3, MSTN, CK activity and miR-1, -133a and miR-206 levels are all significantly different in DE50-MD compared to WT at all timepoints (3, 6, 9, 12, 15 and 18-months). However, there are age-related differences: CCL2, MYOM3 and CK activity all peak in serum at 6-months of age and decline steadily thereafter, while MSTN shows the greatest difference between genotypes between 9-12 months, and the 3 miRs show no significant variation with age. In comparison, the rest of the inflammatory cytokines show greater variation between ages and within genotypes, with significant differences typically seen at 9-months of age for the interleukins, while mean KC-LIKE is elevated at 3, 12, 15 and 18-months of age. We have speculated that the 6-month peak and subsequent decline in MYOM3 and CK activity is a result of a decline in physical activity conducted day-to-day by the dystrophic dogs, as a result of disease progression. In support of this, beyond 6-months, DE50-MD dogs have reduced capacity to withstand eccentric contraction (Riddell \n<i>et <\/i>al., 2023) and, as we have shown in this study, walk significantly shorter 6MWDs. As 6-month-old DE50-MD dogs walk greater absolute and stride-length adjusted distances at 6- compared to 12-months, it is possible that the greater the distance walked the greater the increase in serum markers of muscle damage. Thank you for your suggestions: we have added discussion of the potential influence of age on the exercise-biomarker response test into our manuscript.","URL":"https:\/\/wellcomeopenresearch.org\/articles\/9-681\/v2#referee-comment-6060","order":1,"name":"referee-comment-6060","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.25648.r112850, Ines Barth\u00e9l\u00e9my, Univ Paris-Est Cr\u00e9teil, Cr\u00e9teil, 31 Dec 2024, version 1, 1 approved, 1 approved with reservations","URL":"https:\/\/wellcomeopenresearch.org\/articles\/9-681\/v1#referee-response-112850","order":2,"name":"referee-response-112850","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Dominique Riddell<\/b>; \n<i>Posted: 07 Mar 2025<\/i>; Thank you for feedback and for raising these points. We discuss them below and have adjusted our manuscript accordingly.  \n<b>Summary.&nbsp;<\/b>\n<i>I wonder why the authors chose to perform this biomarker study at 12 months, which is quite late in the life of the dogs in terms of their clinical evolution. At an earlier time point, the CK elevation would probably have been more pronounced, and possibly that of other biomarkers, at least the dystromiRs and inflammatory cytokines. Therefore, I think that the conclusion that MYOM3 and CCL2 are not affected by exercise should be drawn with more caution, because it might be different at earlier time points with a more pronounced exercise-induced CK increase.<\/i> &nbsp;  \n<b>Response:&nbsp;<\/b>Thank you for raising this. We discuss this point specfically below in response to your questions. We have also edited our statements in the abstract and discussion to limit our conclusions to the single timepoint tested in the biomarker-response section of the study. &nbsp;  \n<b>1. <\/b>\n<i>How is the time not walking measured?<\/i> &nbsp;  \n<b>Response:&nbsp;<\/b>Time spent not walking (standing, sitting or lying down) was recorded throughout the test (as time continued to elapse on the stopwatch), with total time not walking combined. We have now clarified this in the methods section. &nbsp;  \n<b>2. <\/b>\n<i>Are the tests filmed and is video analysis used?<\/i> &nbsp;  \n<b>Response:<\/b> For this work no, they were not filmed. However, we have more recently filmed the&nbsp;6MWTs with a view, in future to examining gait and\/or behavioural differences. &nbsp;  \n<b>3.&nbsp;<\/b>\n<i>The 6MWT is highly standardized in humans, in particular the instruction to walk as far as possible. Has the test been performed in a walking gait in dogs? It seems difficult to force dogs to maintain a walking gait, and perhaps a bit artificial, so how was this done?<\/i> &nbsp;  \n<b>Response:&nbsp;<\/b>As you allude to, one of the key challenges when converting the human 6MWT to a canine 6MWT is conveying the instruction to walk as far as possible and achieving cooperation from the dogs. In order to achieve this, the test was standardised as much as possible whilst allowing for encouragement during the test to maintain engagement with the task. Each animal was walked on the handler\u2019s right side, controlled on a leash at the dog\u2019s natural pace around the circuit. Dogs were gently encouraged with voice commands, treats and gentle leash tugs when required but allowed to slow or stop as necessary. We have added detail of our approach to encourage walking at the dog\u2019s natural pace to the methods. All dogs were habituated to the testing area as this was an area they were weighed in twice weekly. As already mentioned in our discussion, 12 week old puppies, regardless of genotype, were easily distracted and this perhaps contributed to the longer time spent not walking in WT dogs at younger ages, whereas more mature dogs walked for a greater percentage of the 6 minutes, suggestive of a greater understanding of what is required of them and their enhanced focus. As the reviewer will no doubt be aware, there are related practical challenges when utilising the 6MWT in young DMD boys and as suggested in the manuscript, direct activity monitoring might help overcome some of the biases or other practical constraints of the 6MWT in dogs. &nbsp; \n<b>4. Do the authors think that cognitive impairments, which they have already described in their model, might affect the performance of DE50-MD dogs in this test?<\/b> &nbsp;  \n<b>Response:&nbsp;<\/b>This is an important point, thank you for raising it. Yes indeed, the cognitive phenotype could contribute to the performance of the DE50-MD dogs in the 6MWT. In our previous work (Crawford \n<i>et al<\/i>., 2022), we showed that, while DE50-MD dogs demonstrate comparable rates of learning new tasks to WT dogs, they show reduced focus on the tasks. This may also occur during the 6MWT: time spent not walking could be due to muscle functional deficits or a loss of focus and engagement with the test. DE50-MD dogs also show reduced exploratory behaviour, which perhaps also contributes to a reduced interest in walking around the 6MWT assessment area. It has also recently been shown that DMD patients with neurodevelopment disorders (including autism and attention-deficit\/hyperactivity disorder) perform significantly worse in motor function tests than those without such disorders \u2013 this was true for a range of timed function tests and the total NSAA score (Keselman \n<i>et al<\/i>., 2025). We have added discussion of these factors to the manuscript. It will be of interest in the future to evaluate some of these aspects in animals that receive a purely muscle-directed treatment in order to try to separate cognitive from musculoskeletal dysfunction, which is otherwise challenging. Crawford AH, Hildyard JCW, Rushing SAM, et al.: Validation of DE50-MD dogs as a model for the brain phenotype of Duchenne muscular dystrophy. \n<i>Dis Model Mech.<\/i> 2022;15(3): dmm049291. 35019137 10.1242\/dmm.049291 8906169 Keselman D, Glanzman A, Thelen MY, Prosser LA, McGuire J, Matesanz SE. Motor function testing rates and outcomes in Duchenne muscular dystrophy with comorbid autism and attention-deficit\/hyperactivity disorder. Neuromuscul Disord. 2025 Jan 30;48:105281. doi: 10.1016\/j.nmd.2025.105281. Epub ahead of print. PMID: 39946777. &nbsp;  \n<b>5. <\/b>\n<i>About the biomarkers.The CK elevation seems mild, but this may be due to the graphical presentation. I would suggest adding a Y-axis cut-off in Figure 5H to make it clearer. It would also be useful to add information about the range of elevation in the results section.<\/i> &nbsp;  \n<b>Response:&nbsp;<\/b>Thank you for this suggestion: we agree that the graphical presentation compresses the elevation: we selected a log-scale in order to visualise both WT and DE50-MD on the same axis as the results for the 2 genotypes are orders of magnitude apart. Having tried several options for cutting the Y-axis we were unable to improve the clarity significantly. Therefore, we have instead added graphs showing the percentage increase pre- to 3-hours-post 6MWT for the 2 biomarkers that showed a significant increase: these graphs clearly demonstrate the CK and KC-LIKE elevation. We have also added information about the range of elevation in the results section. &nbsp;  \n<b>6.&nbsp;<\/b>\n<i>I question your decision to perform this analysis at 12 months of age rather than earlier, when the dogs still have myofibers to break down. The CK increase would probably have been more pronounced and the assessment of the sensitivity of the other biomarkers to exercise would have been more reliable.<\/i> &nbsp;  \n<b>Response:&nbsp;<\/b>We understand the point raised by the reviewer that it would be ideal if&nbsp;we could have examined pre and post exercise blood samples from all dogs at all ages however, a key factor in our decision to conduct the study at this single time point was dog availability and importantly, for ethical reasons (given that this would have very substantially increased the number of times blood was collected from each animal). The study was performed as part of our ongoing natural history work, for which we first established a panel of suitable biomarkers in non-exercised dogs, as well as establishing the utility of the 6MWT itself, before conducting the biomarker response study; however, by this time, all dogs were already recruited and part-way through the study. Hence, we only had sufficient N-numbers of dogs for a well-powered exercise study at 12-months of age. We see the exploration at the 12 month time point as a proof of principle, as our preliminary biomarker work identified the selected markers as highly suitable for differentiating between genotypes in 12-month old dogs. But we agree that it is possible that exercise-induced increases in serum biomarkers with the 6MWT might correlate with the total amount of exercise performed and so it would certainly be interesting to perform this analysis at additional time points, though this would have to be performed in future studies. We have addressed this limitation in the text and have been more cautious with our conclusions&nbsp;in light of our test being restricted to a single timepoint. &nbsp;  \n<b>7. <\/b>\n<i>In the paper published by Acosta&nbsp;et al. in 2016 on the 6 MWT in GRMD dogs and its effect on CK levels, significant change in CK was detected after the 6 MWT at 6 months of age, but not at 12 months of age. This should be included in the limitations of the study in the Discussion section.<\/i> &nbsp;  \n<b>Response:&nbsp;<\/b>As discussed, in contrast to the GRMD work, we did find a significant increase in CK at 12-months, indicative of differences between our models, which is corroborated by our other natural history work and is perhaps unsurprising based on the different breeds and sizes. The DE50-MD percentage increase in CK activity (median: 132%; range 59-326) was closer to the 6-month GRMD dogs (median: 104%; range 93-328) than the 12-months GRMD (median: 18%; range -0.4-67.9), despite the 12-month-old DE50-MD and GRMD dogs both walking shorter distances than the 6-month GRMD dogs. Perhaps, had we also performed the biomarker-response study at 6-months, when DE50-MD were capable of walking greater distances, we might have seen a greater CK increase than at 12-months. As above, we agree this is a limitation and have added to the discussion, with reference to the Acosta paper.","URL":"https:\/\/wellcomeopenresearch.org\/articles\/9-681\/v2#referee-comment-6061","order":3,"name":"referee-comment-6061","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.26410.r121945, Yuri van der Burgt, Leiden University Medical Center, Leiden, 24 Apr 2025, version 2, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/9-681\/v2#referee-response-121945","order":4,"name":"referee-response-121945","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Dominique Riddell<\/b>; \n<i>Posted: 06 Jun 2025<\/i>; \n<b><i>In this manuscript the authors report on the evaluation and applicability of a six-minute walk test in the DE50-MD canine model of Duchenne muscular dystrophy (DMD). The study is well-presented and the report is sound and clear. Although the scientific quality of this work is very much appreciated, it may not be obvious how this study will contribute to a more clinical aim to improve DMD patient care. <\/i><\/b> \n<b><i>Clearly the use of an animal model adds to our knowledge of the disease and as the authors write \u201ccanine models are invaluable for preclinical evaluation of treatments\u201d. The value of using a canine model is undisputed, but the concerns of this reviewer relate to whether a walk test is suitable for evaluations of the animal model.<\/i><\/b> \n<b><i>In the abstract the authors correctly state that&nbsp; \u201cThe 6-minute walk test (6MWT) is a key functional outcome measure for DMD patient clinical trials\u201d, but why a similar walk test the desired way-to-go for evaluation of the canine model? This reviewer agrees with a concern raised by a previous reviewer: \u201cThe 6MWT is highly standardized in humans, in particular the instruction to walk as far as possible\u2026 It seems difficult to force dogs to maintain a walking gait, and perhaps a bit artificial, so how was this done?\u201d. <\/i><\/b> \n<b>Response:<\/b> The 6MWT is in fact widely used in the veterinary arena and has been used previously in the Golden Retriever muscular dystrophy (GRMD) model of DMD. See for example:  Boddy KN, Roche BM, Schwartz DS, Nakayama T, Hamlin RL. Evaluation of the six-minute walk test in dogs. Am J Vet Res. 2004 Mar;65(3):311-3. doi: 10.2460\/ajvr.2004.65.311. PMID: 15027678. Swimmer RA, Rozanski EA. Evaluation of the 6-minute walk test in pet dogs. J Vet Intern Med. 2011 Mar-Apr;25(2):405-6. doi: 10.1111\/j.1939-1676.2011.0689.x. Epub 2011 Feb 25. PMID: 21352372; PMCID: PMC3052904. Acosta AR, Van Wie E, Stoughton WB, Bettis AK, Barnett HH, LaBrie NR, Balog-Alvarez CJ, Nghiem PP, Cummings KJ, Kornegay JN. Use of the six-minute walk test to characterize golden retriever muscular dystrophy. Neuromuscul Disord. 2016 Dec;26(12):865-872. doi: 10.1016\/j.nmd.2016.09.024. Epub 2016 Oct 3. PMID: 27818009. Gallman J, Lee-Fowler T, Clark-Price S, Grobman M. Evaluation of infrared thermography and 6-minute walk tests to assess airflow limitation, impaired thermoregulation, and exercise intolerance in dogs with brachycephalic obstructive airway syndrome. PLoS One. 2023 Mar 31;18(3):e0283807. doi: 10.1371\/journal.pone.0283807. PMID: 37000798; PMCID: PMC10065248.  The primary aim of this study was to assess the suitability of the 6MWT as a biomarker for use in future pre-clinical trials with this particular canine model of DMD. While other biomarkers such as circulating molecular markers may be argued to offer greater objectivity, they often do not correlate with muscle function, a primary clinical outcome measure for DMD treatments that aim to combat the progressive muscle degeneration and atrophy that characterises the disease. For this reason, it is essential that we have assessments of muscle function in our pre-clinical trials. Indeed, that is what regulatory authorities require. Please refer to our response to Ines&nbsp;Barth&eacute;l&eacute;my\u2019s\n<b> <\/b>comment regarding how we designed the protocol to standardise the method. This was based on standardised testing procedures currently used in dogs.  \n<b><i>The authors are encouraged to work on more objective parameters, such as molecular biomarkers. Indeed they include work on such markers, but unfortunately the focus lies on whether or not such biomarkers change as a results of exercise, in this case the walk test itself (\u2026). It is recommended to perform the work on the identification and development of biomarkers that can replace a subjective walk test (that varies day-to-day with ability and motivation). <\/i><\/b> \n<b>Response:<\/b> We refer the reviewer to our already established, extensive and varied panel of objective biomarkers, clearly detailed in the manuscript, including circulating biomarkers, histological markers of muscle disease, MRI quantification of muscle volumes and composition, RT-qPCR for expression of many DMD-related genes, cardiac MRI, ECG parameters, and cognitive testing. We have also used additional methods of assessing muscle function. These include muscle physiology testing under general anaesthesia via nerve stimulation. Whilst potentially more objective, these assessments must be carried out under general anaesthesia and the manner of muscle stimulation is distinct from that which occurs naturally. We have also established an activity monitoring protocol to quantify natural daily activity over a prolonged period of time (Karimjee \n<i>et al<\/i>. 2024, Karimjee \n<i>et al<\/i>. 2025 ). The 6MWT compliments these measures and adds values as a straightforward, inexpensive test, examining natural muscle use, that can be conducted more frequently and non-invasively, and which is utilised in DMD patients and other dog models giving potential for cross-study comparisons. Regarding the potential for day-to-day variation in motivation and ability, we showed in this paper that walk distance was very consistent, with no significant difference between walk 1 and walk 2, giving confidence in the repeatability of the test on different days.  \n<b><i>Moreover, it is not clear why we should measure such markers after the walk test (\u201cThere is therefore a need for identification of serum biomarkers that are less susceptible to exercise-induced fluctuations.\u201d and \u201cThe effect of exercise on the circulating levels of these biomarkers is yet to be determined.\u201d). Instead of evaluating the effect of exercise on the markers these should rather be used to monitor therapeutic interventions and measured in a longitudinal manner. <\/i><\/b> \n<b>Response:<\/b> We have already extensively evaluated and established a large panel of objective biomarkers assessed in a longitudinal manner, over the typical timecourse required for therapeutic interventions (0-18 months), as referenced in the manuscript and in response to the previous comment. In terms of serum biomarkers, we have published two papers on our establishment of longitudinal circulating biomarkers for future monitoring therapeutic interventions, including those biomarkers presented in this study. Our follow-up, and important question, addressed in this paper was whether exercise might influence these biomarkers, thus rendering their concentrations subject to variation dependent on recent activity. When monitoring responses to therapeutic interventions we require biomarkers that are robust to such perturbations in order to distinguish a true treatment-induced effect on biomarker concentration from an exercise-induced effect. &nbsp;  \n<b><i>The authors conclude that \u201cThe 6MWT effectively discriminates DE50-MD from WT dogs aged 3-18 months and a single 6MWT is sufficient for future studies.\u201d, but such a differentiation is not the purpose of the walk test. Note that 6MWT is meant for disease monitoring rather than diagnostic purposes. This comment also affects the title that should rather read: \u201cEvaluation of a six-minute walk test and circulating biomarkers in the DE50-MD canine model of Duchenne muscular dystrophy\u201d. A less important comment concerns the term \u201cblood-borne biomarker\u201d. Although this reviewer acknowledges not to be a native speaker, the term blood-borne is more common for infectious diseases and moreover misleading. Please just refer to these markers as \u201cblood-based biomarkers\u201d or \u201ccirculating biomarkers\u201d.<\/i><\/b> \n<b><i>&nbsp;<\/i><\/b> \n<b>Response:<\/b> Before using the 6MWT as a biomarker it was essential to assess its ability to differentiate between the 2 genotypes with sufficient power to allow detection of improvements following an intervention. Having achieved that in this paper, we can use this biomarker to assess the impact of therapies on muscle function. Regarding the title, stating that we are assessing the impact of exercise on the circulating biomarkers rather than simply assessing circulating biomarkers is an important distinction; it shows that this work is adding to, rather than repeating, work already performed in this model of DMD.","URL":"https:\/\/wellcomeopenresearch.org\/articles\/9-681\/v2#referee-comment-6143","order":5,"name":"referee-comment-6143","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was supported by The Wellcome Trust (101550). Dr Riddell\u2019s salary was partly supported by Pfizer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":6,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}