Enthalpy efficiency of the soleus muscle contributes to improvements in running economy

During human running, the soleus, as the main plantar flexor muscle, generates the majority of the mechanical work through active shortening. The fraction of chemical energy that is converted into muscular work (enthalpy efficiency) depends on the muscle shortening velocity. Here, we investigated the soleus muscle fascicle behaviour during running with respect to the enthalpy efficiency as a mechanism that could contribute to improvements in running economy after exercise-induced increases of plantar flexor strength and Achilles tendon (AT) stiffness. Using a controlled longitudinal study design (n = 23) featuring a specific 14-week muscle–tendon training, increases in muscle strength (10%) and tendon stiffness (31%) and reduced metabolic cost of running (4%) were found only in the intervention group (n = 13, p < 0.05). Following training, the soleus fascicles operated at higher enthalpy efficiency during the phase of muscle–tendon unit (MTU) lengthening (15%) and in average over stance (7%, p < 0.05). Thus, improvements in energetic cost following increases in plantar flexor strength and AT stiffness seem attributed to increased enthalpy efficiency of the operating soleus muscle. The results further imply that the soleus energy production in the first part of stance, when the MTU is lengthening, may be crucial for the overall metabolic energy cost of running.


Comments to the Author
In this study, the authors examined the effects of a resistance training program on running economy, and additionally examined how changes in running economy were associated with changes in estimated soleus muscle strength, Achilles tendon stiffness, and operating soleus muscle efficiency, force-length, and force-velocity behaviour. This study provides insight into the mechanisms that may underly improvements in running economy with resistance training. The majority of our understanding of the role of series elasticity on efficiency is from controlled in situ or simulation studies. Thus, this study also provides novel insight into the implications of in vivo muscle and tendon properties during real-world conditions. This manuscript is well-written and interesting to read, and the methods appear sound and appropriate for addressing the research questions. I only have a few comments below that aim to clarify details of the methodology and interpretation of the results. Comments: 1. Lines 137-144: Given that increased plantar flexor strength and tendon stiffness are identified as a possible mechanism underlying the main results of this paper, it would be helpful to provide further details of how these variables were measured rather than referring readers to other papers. For example, in Supplementary material 1, section 2: "Furthermore, the contribution of the antagonistic muscles to the ankle joint moment was considered by means of an EMG-based method [4]." What specific method was this? "which was determined using the tendon-excursion method [5,6] and corrected for tendon alignment during the contraction [7]." How were the moment arms corrected for tendon alignment? "The MTJ displacement artefacts due to an unavoidable change in the ankle joint angle during the MVCs was corrected [8] and the five contractions were averaged to give a reliable measure of the elongation [9]. The AT stiffness was calculated between 50% and 100% of the maximum tendon force using linear regression [10]" How were the changes in ankle joint angle corrected? Currently the reader would have to consult a range of other papers to fully understand the methods and their justification. More details of these methods and less reliance on previous works would be beneficial.
2. Similar to 1., given that running economy is an important variable in this paper, further details in the main text would be helpful. Since the section "Energetic cost of running" in supplementary material 1 is only one paragraph long, could this not be included in the methods section of the main text? I realize the authors may be limited in terms of length; however, these details are important for interpreting the results of this paper. Similarly, at least the first paragraph of the section "Statistics" in supplementary material 1 could be included in the main text. Important methods that could affect interpretation of results and conclusions should be easy for readers to access in the main text.
3. Line 194: Why did the authors use an efficiency-velocity function rather than a more established metabolic power function (e.g. Minetti & Alexander, 1997 or Umberger, 2010, etc.)? Mechanical work and metabolic cost depend on factors other than just velocity, so why is an efficiency function that depends only on velocity, instead of separately estimating mechanical work and metabolic cost that depend on muscle velocity, length, activation, etc., appropriate for this study? Further explanation/justification in the text would be helpful. Also, the fitted values in Table 1 of Hill (1967) are for frog muscle at 0 degrees C. Since frogs are ectotherms, the muscle temperature would be near that of the external environment, far below physiological temperature for human muscle. This could affect both muscle force and velocity (see James, 2013 for review) and therefore the fitted function. Additionally, amphibian muscle contains larger concentrations of parvalbumin compared to terrestrial muscles, which can alter the heat rate and estimated metabolic cost ( 4. Line 268: "… the results provide additional evidence that a combination of greater plantar flexor muscle strength and Achilles tendon stiffness decrease the energy cost of running [14,15] and indicate that the soleus enthalpy efficiency is a contributive determinant." It's alluded to with "a combination" but consider an additional sentence here noting that an increase in stiffness by itself may not increase efficiency. Later in line 349 the authors state "strength increases without concomitant stiffening of the AT after a period of training may increase levels of operating and maximum AT strain [24], which have been associated with pathologies [53] but also possible functional decline [54]." Function may also decline with increases in stiffness without concomitant increases in muscle strength. For example, see Figure 5 in Lichtwark and Wilson (2005) in which muscle efficiency during running decreased with increases in AT stiffness beyond the optimal stiffness. 5. Line 291: "The exercise-induced increase in muscle strength and AT stiffness resulted in an alteration of the operating fascicle velocity profile that led to a significant increase of the enthalpy efficiency of the operating soleus […], improving the efficiency of muscular work production." Because the only factor that was manipulated in this study was the exercise intervention, changes in muscle strength, AT stiffness, fascicle velocities, and enthalpy efficiency are only associated with one another rather than there being any causal relationship between them.

07-Dec-2020
Dear Dr Bohm: Your manuscript has now been peer reviewed and the reviews have been assessed by an Associate Editor. The reviewers' comments (not including confidential comments to the Editor) and the comments from the Associate Editor are included at the end of this email for your reference. As you will see, the reviewers and the Editors have raised some concerns with your manuscript and we would like to invite you to revise your manuscript to address them.
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Best wishes, Dr John Hutchinson, Editor mailto: proceedingsb@royalsociety.org Associate Editor Board Member: 1 Comments to Author: Dear Dr. Bohm, Thank you for submitting your manuscript entitled "Enthalpy efficiency of the soleus muscle contributes to improvements in running economy" to the Proceedings of the Royal Society. I have received two peer reviews, and both are highly supportive of your manuscript but also have a several suggestions, which I hope you will find useful when revising your manuscript. Proceedings B aims to publish studies that significantly increase or alter our current understandings in a way that is relevant to a broad readership beyond the disciplinary area of the manuscript. Both reviewers find your study of high scientific importance and broad interest, and many of their comments aim mainly at improving the clarity of the manuscript. The reviewers furthermore ask for additional information on the methodology and share their thoughts concerning the findings, cautioning against overstatements and arguing for nuance.

Reviewer(s)' Comments to Author:
Referee: 1 Comments to the Author(s) General comments: Many thanks for the invitation to review this paper, it was a thoroughly enjoyable and fascinating read. The paper describes the results of a 14-week muscle-tendon strength training intervention that found enhancements in running economy, plantar flexion strength, and Achilles stiffness compared to a control group. An improvement in enthalpy efficiency of the soleus muscle reveals novel insight into the mechanism by which strength training may have a positive effect of the metabolic cost of running.
In my opinion, this study is much needed in this area of research. Papers have speculated in the past around the mechanisms of change associated with improved running economy following a strength training intervention (e.g. Fletcher and Macintosh, 2017, doi: 10.3389/fphys.2017.00433; Blagrove et al., 2018, doi: 10.1007/s40279-017-0835-7), however measuring changes to the intrinsic behaviour of muscles is difficult. This study makes a very good attempt at providing that insight for the soleus. I have some minor comments that I hope will improve clarity and readability of the paper, but overall, I feel that this paper will be of considerable interest to both scientists and applied practitioners.

Specific comments:
Keywords: These should be different to the terms in the study title to enable wider search returns. Please amend. Can I suggest 'calf' 'triceps surae' 'endurance running' 'strength training'?
Line 44: 'for' should read 'in' Line 105: Was allocation to groups completely random or were participants matched for running economy and randomised by matched pairs (or similar) to ensure minimal differences existed between groups at baseline?
Line 106: Was the participants only sport/exercise running? It would be useful for others (particularly those undertaking reviews and meta-analyses in this area) to be able to accurately determine if participants were trained 'runners' or simply people that ran as a small part of a wider exercise/sport training routine.
Line 106: Please define 'severe' in brackets here (i.e. days/weeks away from running with injury) Line 108: Why were only rear-foot striking runners considered?
In female participants, was the menstrual cycle accounted for or hormonal contraceptive use during recruitment and testing?
A criticism often levelled at studies in the area of strength training for endurance athletes is that studies rarely equate the total amount of physical exercise done between groups, i.e. the control group do not have 'placebo' exercise(s) or add running training to match the duration of strength work performed by the intervention group (e.g. Dankel et al., 2017, doi: 10.1080/02640414.2017.1398884). Although a performance measure was not taken in this study, how do the authors know that the change in running economy they observed is not due to differences in the amount of physical training performed? An alternative, in practice, for runners could be to add running training instead of strength training to their routine, which may produce even larger improvements in economy.
The changes in soleus fascicle behaviour were not quantified in the control group. I am slightly puzzled why not. Would the authors consider this a limitation of the study?
Exercise protocol: Given that a single strength training exercise was used in the intervention I would strongly recommend that authors include an image of the exercise apparatus and set-up. I appreciate there are currently a high number of figures included but I would contend this is important for both scientific replication and applied practice.
Line 147: Why was 2.5 m/s used as the speed for all participants? Was this sufficiently slow enough to ensure a plateau in oxygen consumption and RER value of <1 during the collection period?
Line 149: The citation here is a paper comparing methods of quantifying energy cost of running. It is not clear which method was used without referring to the supplementary material.
Line 205: Which post-hoc adjustment was used?
Line 210: How were the effect sizes interpreted?
Line 229: There appears to be a word missing in this sentence. 'an altered lengthening-shortening behaviour' or similar Line 272: It would be more accurate to discuss the change in economy in the context of withinparticipant variability (measurement error), rather than between-participant variability, which depends on the sample. A subtle tweak to wording and the reference (eg Blagrove et  Line 304-305: Why does the higher maximum plantar flexion moment indicate hypertrophy has occurred? It would be unusual to expect substantial hypertrophy with short-duration isometric contractions. Why can the improvements in strength not be explained as neural adaptation? If so, the discussion below this statement will need to be amended.
Line 346: 'a' seems to be a typographical error here. Line 350: There appears to be a word missing between 'training' and 'may' Line 355: 'endurance performance' should read 'running economy' here as no performance measures were taken.
It has long been recognised that the soleus possesses a high proportion of slow twitch muscle fibres compared to other muscle groups (eg Gollnick et al., 1974, doi: 10.1007/BF00587415). Clearly it is possible to make the soleus stronger and given its role in locomotion and energy cost during exercise, it would certainly make sense for runners to strengthen the muscle. However, do authors think that the soleus has a limited capacity to improve its maximal force output due to its morphological characteristics? The intervention applied here would certainly be novel for the participants, thus beneficial, but would long-term engagement with this type of training for soleus continue to yield benefits in running economy?
Referee: 2 Comments to the Author(s) In this study, the authors examined the effects of a resistance training program on running economy, and additionally examined how changes in running economy were associated with changes in estimated soleus muscle strength, Achilles tendon stiffness, and operating soleus muscle efficiency, force-length, and force-velocity behaviour. This study provides insight into the mechanisms that may underly improvements in running economy with resistance training. The majority of our understanding of the role of series elasticity on efficiency is from controlled in situ or simulation studies. Thus, this study also provides novel insight into the implications of in vivo muscle and tendon properties during real-world conditions. This manuscript is well-written and interesting to read, and the methods appear sound and appropriate for addressing the research questions. I only have a few comments below that aim to clarify details of the methodology and interpretation of the results. Comments: 1. Lines 137-144: Given that increased plantar flexor strength and tendon stiffness are identified as a possible mechanism underlying the main results of this paper, it would be helpful to provide further details of how these variables were measured rather than referring readers to other papers. For example, in Supplementary material 1, section 2: "Furthermore, the contribution of the antagonistic muscles to the ankle joint moment was considered by means of an EMG-based method [4]." What specific method was this? "which was determined using the tendon-excursion method [5,6] and corrected for tendon alignment during the contraction [7]." How were the moment arms corrected for tendon alignment? "The MTJ displacement artefacts due to an unavoidable change in the ankle joint angle during the MVCs was corrected [8] and the five contractions were averaged to give a reliable measure of the elongation [9]. The AT stiffness was calculated between 50% and 100% of the maximum tendon force using linear regression [10]" How were the changes in ankle joint angle corrected? Currently the reader would have to consult a range of other papers to fully understand the methods and their justification. More details of these methods and less reliance on previous works would be beneficial.
2. Similar to 1., given that running economy is an important variable in this paper, further details in the main text would be helpful. Since the section "Energetic cost of running" in supplementary material 1 is only one paragraph long, could this not be included in the methods section of the main text? I realize the authors may be limited in terms of length; however, these details are important for interpreting the results of this paper. Similarly, at least the first paragraph of the section "Statistics" in supplementary material 1 could be included in the main text. Important methods that could affect interpretation of results and conclusions should be easy for readers to access in the main text.
3. Line 194: Why did the authors use an efficiency-velocity function rather than a more established metabolic power function (e.g. Minetti & Alexander, 1997 or Umberger, 2010, etc.)? Mechanical work and metabolic cost depend on factors other than just velocity, so why is an efficiency function that depends only on velocity, instead of separately estimating mechanical work and metabolic cost that depend on muscle velocity, length, activation, etc., appropriate for this study? Further explanation/justification in the text would be helpful. Also, the fitted values in Table 1 of Hill (1967) are for frog muscle at 0 degrees C. Since frogs are ectotherms, the muscle temperature would be near that of the external environment, far below physiological temperature for human muscle. This could affect both muscle force and velocity (see James, 2013 for review) and therefore the fitted function. Additionally, amphibian muscle contains larger concentrations of parvalbumin compared to terrestrial muscles, which can alter the heat rate and estimated metabolic cost (Woledge et al., 1985, pp. 257-260). What are the implications of these considerations on the results of this study?

Line 291: "
The exercise-induced increase in muscle strength and AT stiffness resulted in an alteration of the operating fascicle velocity profile that led to a significant increase of the enthalpy efficiency of the operating soleus […], improving the efficiency of muscular work production." Because the only factor that was manipulated in this study was the exercise intervention, changes in muscle strength, AT stiffness, fascicle velocities, and enthalpy efficiency are only associated with one another rather than there being any causal relationship between them.

Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No
It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Do you have any ethical concerns with this paper? No
Comments to the Author Many thanks for taking the time to provide clear and comprehensive responses to my comments and questions. I am satisfied they have been appropriately addressed. I look forward to seeing this paper published and will circulate it to my networks. I'm sure i'll refer to it regularly.

05-Jan-2021
Dear Dr Bohm I am pleased to inform you that your manuscript entitled "Enthalpy efficiency of the soleus muscle contributes to improvements in running economy" has been accepted for publication in Proceedings B. Congratulations!! You can expect to receive a proof of your article from our Production office in due course, please check your spam filter if you do not receive it. PLEASE NOTE: you will be given the exact page length of your paper which may be different from the estimation from Editorial and you may be asked to reduce your paper if it goes over the 10 page limit.
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Comment:
Many thanks for the invitation to review this paper, it was a thoroughly enjoyable and fascinating read. The paper describes the results of a 14-week muscle-tendon strength training intervention that found enhancements in running economy, plantar flexion strength, and Achilles stiffness compared to a control group. An improvement in enthalpy efficiency of the soleus muscle reveals novel insight into the mechanism by which strength training may have a positive effect of the metabolic cost of running. In my opinion, this study is much needed in this area of research. Papers have speculated in the past around the mechanisms of change associated with improved running economy following a strength training intervention (e.g. Fletcher and Macintosh, 2017, doi: 10.3389/fphys.2017.00433; Blagrove et al., 2018, doi: 10.1007/s40279-017-0835-7), however measuring changes to the intrinsic behaviour of muscles is difficult. This study makes a very good attempt at providing that insight for the soleus. I have some minor comments that I hope will improve clarity and readability of the paper, but overall, I feel that this paper will be of considerable interest to both scientists and applied practitioners.

Response:
Thank you for your thorough and valuable comments.
Specific comments:

Comment:
Keywords: These should be different to the terms in the study title to enable wider search returns. Please amend. Can I suggest 'calf' 'triceps surae' 'endurance running' 'strength training'?

Response:
Thanks for this comment. We replaced four of the keywords to: "Force-length and forcevelocity relationship, enthalpy-velocity relationship, triceps surae, endurance running, strength training, tendon stiffness"

Response:
Corrected. Thank you.

Comment:
Line 105: Was allocation to groups completely random or were participants matched for running economy and randomised by matched pairs (or similar) to ensure minimal differences existed between groups at baseline?

Response:
It was completely random and the outcome parameters for the group comparison were not significantly different at the baseline level as reported.

Comment:
Line 106: Was the participants only sport/exercise running? It would be useful for others (particularly those undertaking reviews and meta-analyses in this area) to be able to accurately determine if participants were trained 'runners' or simply people that ran as a small part of a wider exercise/sport training routine.

Response:
Participants were running on a regular, yet recreational basis with a minimum of twice a week set as an inclusion criterion. None of the participants was involved in professional competitive running. We added to the following information to be more clear (page: 3, line: 103): "Inclusion criteria were age 20 to 40 years, at least two running sessions weekly on a recreational basis and no muscular-tendinous injuries in the previous year."

Comment:
Line 106: Please define 'severe' in brackets here (i.e. days/weeks away from running with injury)

Response:
'Severe' in the context meant potential injuries that affected the running habits of the participants in the past year. We deleted the word to avoid confusion.

Comment:
Line 108: Why were only rear-foot striking runners considered?

Response:
There is an ongoing debate on the effect on foot strike pattern on running economy in the scientific community. Thus, to avoid any potential confounding effects on our study outcomes we excluded this factor by recruiting a homogenous group of rear foot runners, which is also by far the most common strike pattern (1,2,3,4). We added to the following information to be more clear (page: 3, line: 108): "Only habitual rearfoot-striking runners were considered because it is the most common foot strike pattern (4) and also to avoid potential confounding effects of the strike pattern on our study outcomes.

Comment:
In female participants, was the menstrual cycle accounted for or hormonal contraceptive use during recruitment and testing?

Response:
Menstrual cycle status was not considered systematically because of the difficulty to align pre/post measurements sessions and menstrual cycle time points. However, three of the four females of the 13 participants of the intervention group self-reported their cycle status; female 1: pre: early follicular phase (day 3 of cycle) and post: late luteal phase (day 26), female 2: pre: late follicular phase (day 9) and post: early luteal phase (day 16) and female 3: pre: late luteal (day 26) and post: late follicular phase (day 9). Over the time course of the menstrual cycle only the mid-luteal phase has been reported to impair running economy (2,3). Note that none of the three females reported this particular phase of the menstrual cycle during the test sessions. With regard to contraceptives, from the four females only two used a hormone spiral as contraceptive. Such low-dose contraceptives have been suggested to not interfere significantly with running economy (1). However, the specific application of hormone spirals in the context of running economy seems not investigated well to the best of our knowledge. Furthermore, when performing a sensitivity analysis of our reported intervention effect by changing the post energy cost values of the four females, we found that the intervention effect on the energetic cost would remain significant (p < 0.05) in case of up to a ~4% higher post value in all of the four females at the same time. Therefore, considering a) the low number of females in the intervention group, b) that none of the females reported critical mid-luteal phase during testing, c) only two used low-dose contraceptives and d) a quite robust intervention effect against the unlikely case that all the four females would have a higher energetic cost during the post test, we can argue that our found improvement in running economy following training is likely not affected by these factors. Although a performance measure was not taken in this study, how do the authors know that the change in running economy they observed is not due to differences in the amount of physical training performed? An alternative, in practice, for runners could be to add running training instead of strength training to their routine, which may produce even larger improvements in economy.

Response:
Thank you for this important comment. We did not include an additional group that performed a time-matched running training in our experimental design. The reason was that in an earlier study (1) we applied specifically running training (i.e. focusing on the alteration of the running technique) in a group of experienced runners and we did not find any effects on running economy after the 14 weeks of running training (3x/week 30min). Furthermore, several studies in the past have shown that running training itself does not improve running economy (ref. 2: 6 weeks, ~160 km,) particularly in trained runners as in our study (ref. 3: 8 weeks, 210 km added). Therefore, we are confident that the specific strength training, which improved muscle strength of the plantar flexors and Achilles tendon stiffness, was the reason for the improved running economy and that running training in experienced runners cannot cause any additional improvements in running economy.

Comment:
Exercise protocol: Given that a single strength training exercise was used in the intervention I would strongly recommend that authors include an image of the exercise apparatus and set-up. I appreciate there are currently a high number of figures included but I would contend this is important for both scientific replication and applied practice.

Response:
Thanks for this comment. We added the following figure including a descriptive caption in the revised version of the manuscript. Please note that it has been placed in the supplementary material due to the limited space available.

Comment:
Line 147: Why was 2.5 m/s used as the speed for all participants? Was this sufficiently slow enough to ensure a plateau in oxygen consumption and RER value of <1 during the collection period?

Response:
A running velocity of 2.5 m/s was used in order to ensure that all participants ran at steady-state, which is a key aspect for the assessment of running economy. The plateau of the oxygen consumption was visually confirmed for each individual and trial and a representative example curve is given in the supplementary material. The average RER for the control group was pre 0.89 ± 0.05 and post 0.87 ± 0.13 and for the intervention group pre 0.94 ± 0.04 and post 0.95 ± 0.05, respectively. We added to the following sentence to be more clear in the revised manuscript (page: 4, line: 145): "During an 8-minute running trial on a treadmill at 2.5 m/s, expired gas analysis was conducted and rate of oxygen consumption (V̇O2 ) and carbon dioxide production (V̇CO2) was calculated as average of the last three minutes [15]. Running economy was then expressed in units of energy [4,30]

Comment:
Line 149: The citation here is a paper comparing methods of quantifying energy cost of running. It is not clear which method was used without referring to the supplementary material.

Response:
Many apologies. The cited study investigated the appropriateness of the used formula and this was the reason for inserting only this reference. In the revised manuscript, we added the original study and also presented the formula in the main text (see previous comment). Please note that several detailed information is presented in the supplementary material because of the limited space in the main text.

Comment:
Line 205: Which post-hoc adjustment was used?

Response:
A Benjamini-Hochberg correction was applied and adjusted p-values are reported. This information can be found in the supplementary material (section statistics).

Comment:
Line 210: How were the effect sizes interpreted?

Response:
Effect sizes were interpreted according to Cohen 1988, were 0.2 ≤ g < 0.5 indicates a small, 0.5 ≤ g < 0.8 indicates a medium, and g ≥ 0.8 indicates a large effect size. This information can be found in the supplementary material (section statistics).

Comment:
Line 229: There appears to be a word missing in this sentence. 'an altered lengtheningshortening behaviour' or similar

Response:
Thank you for this comment. What we intended to say here is a general description of the MTU behavior that does not refer to intervention effects. Therefore, the behavior is not "altered". We think that this solves a language issue.

Comment:
Line 272: It would be more accurate to discuss the change in economy in the context of within-participant variability (measurement error), rather than between-participant variability, which depends on the sample. A subtle tweak to wording and the reference (eg Blagrove et

Response:
Thank you for this valuable comment. The sentence was changed accordingly in the revised manuscript and one mentioned references were added (page: 7, line: 262): "Following the intervention, the energetic cost of running was significantly reduced by about 4%, a quantity reported to be above test-retest typical errors [38] and to substantially enhance endurance running performance [39]."

Comment:
Line 304-305: Why does the higher maximum plantar flexion moment indicate hypertrophy has occurred? It would be unusual to expect substantial hypertrophy with short-duration isometric contractions. Why can the improvements in strength not be explained as neural adaptation? If so, the discussion below this statement will need to be amended.

Response:
We agree with the reviewer that neural adaptation could have contributed to the obtained strength gains following training besides hypertrophy. While there is evidence that neural adaptations may precede morphological responses during the early weeks of strength training onset (1), the intervention duration of our study was quite long at 14 weeks. Several studies have shown an increasing contribution of morphological changes (hypertrophy) following the first 5-6 weeks of training (2,3) beyond neural adaptations (2,5). Moreover, strength training using explicitly isometric contractions have been shown to provide a sufficient stimulus to induce muscle hypertrophy (6,7). In our study, EMGmax obtained during the maximum voluntary plantar flexions was not changed following the training (pre 0.409 ± 0.114 mV and post 0.410 ± 0.092 mV, p = 0.300). Similarly, the training had no effect on the antagonistic co-activation (tibialis anterior EMG 0.034 ± 0.016 mV and post 0.034 ± 0.013 mV, p = 0.923). Taken together, the absence of changes in these parameters may not exclude it on other structural levels but strongly indicate that neural aspects may not the primary course of the found strength gains after the 14 weeks of training.
According to the reviewers comment we changes our formulation in the revised manuscript (page: 8, line: 293): "However, the higher maximum plantar flexion moment along with no significant changes in EMGmax during the MVCs (pre 0.409 ± 0.114 mV and post 0.410 ± 0.092 mV, p = 0.300) and antagonistic coactivation (tibialis anterior EMG 0.034 ± 0.016 mV and post 0.034 ± 0.013 mV, p = 0.923) as measures for neural adaption after training strongly indicate muscle hypertrophy, resulting in a 13% increase of Fmax (pre 2903 ± 750 N, post 3285 ± 831 N)."

Comment:
It has long been recognised that the soleus possesses a high proportion of slow twitch muscle fibres compared to other muscle groups (eg Gollnick et al., 1974, doi: 10.1007/BF00587415). Clearly it is possible to make the soleus stronger and given its role in locomotion and energy cost during exercise, it would certainly make sense for runners to strengthen the muscle. However, do authors think that the soleus has a limited capacity to improve its maximal force output due to its morphological characteristics? The intervention applied here would certainly be novel for the participants, thus beneficial, but would long-term engagement with this type of training for soleus continue to yield benefits in running economy?

Response:
Thank you for this important comment. There are reports that fast-twitch fibres feature a greater hypertrophic response to resistance training compared to slow-twitch fibres (1,2,3). Therefore, one might suggest that the soleus muscle is limited in its capacity to improve its maximal force following training. However, the findings are inconsistent and there are studies reporting similar training-induced hypertrophy in slow and fast-twitch fibres (4,5). Therefore, we can argue that the morphological characteristics of the soleus muscle might not be the limiting factor. However, based on the present study we can conclude that two mechanisms contribute to the advantageous work generation by soleus, i.e. the operating enthalpy efficiency and operating force-length potential. The force-length potential was already high throughout the entire stance phase both before and after the training intervention (pre 0.89%, post 0.88%). The enthalpy efficiency throughout the stance was influenced by the intervention and increased by 7% to 92% of the maximum efficiency. Thus, the potential available adaptation range of the enthalpy efficiency for further improvements due to prolonged training seems to be the limiting factor.