Extra Protein = Only Marginal Extra-Gains, No Special Effect on Muscle Architecture | Plus: Blend Beats Whey, Again

No, the message of this article is not that protein shakes don't work. It is that your (hopefully) tasty 20g of serving of whey is not going to build slabs of extra muscle.

You all know studies which show that protein supplementation during resistance exercise training enhances muscle hypertrophy. As a SuppVersity reader, you will yet also be aware of the numerous studies which indicate that extra-protein (before or after workouts) can be wasted if the baseline protein intake of the subjects amounts to 1.2-1.5g/kg protein, already (cf Table 1).

For some of you, this is yet probably not the only surprise this article holds. The large-scale clinical trial by Reidy, et al. did after all also confirm that protein blends may yield slightly better results than everyone's beloved whey protein.

High-protein diets are much safer than some 'experts' say, but there are things to consider...

Practical Protein Oxidation 101

5x More Than the FDA Allows!

Native Whey = Extra Gainz?

Satiety: Casein > Whey? Wrong!

Protein Timing DOES Matter!

High Protein not a Health Threat

In their large-scale clinical trial (cf. Reidy 2016a,b), the scientists from Texas broadened the scope of their analysis of the effects of a 12-week resistance training program from mere changes in body composition to measures of individual and myofiber-specific cross-sectional area increases, satellite cell numbers and the extent to which the muscle domains multiplied by myonuclear addition (that's important for continuous gains).

Figure 1: CONSORT (Consolidated Standards of Reporting Trials) diagram of study recruitment, enrollment, randomization follow-up, and analysis (from Reidy 2016b).

To this ends, Reidy et al. recruited 70 subjects (of which 58 completed the study;  the participants were healthy and recreationally active but were not engaged in any regular exercise-training program in form of <2 sessions high-intensity aerobic or resistance exercise/wk at the time of enrollment) who participated in a supervised whole body progressive resistance training program thrice a week for 12 weeks. With a habitual protein intake of ~1.3g/kg body weight, all subjects were "in the zone", i.e. within the perimeter of which previous studies show that it's enough to reduce, if not nullify the benefits of post-workout protein supplementation.

Figure 2: Schematic of the resistance exercise training protocol (Reidy. 2016b)

All subjects underwent the same standardized resistance training program (see Figure 2): All exercise training sessions were performed under supervision and on non-consecutive days, 3 times weekly, with 4 rest days per week.

"RET was performed at an intensity of 60-80% of 1-repetition maximum (1-RM) and consisted of 3-4 sets of 8-10 repetitions performed to technical failure during the last set for each exercise.  In week 1, 3 sessions were conducted at 3 sets of 10 repetitions at 60% 1-RM.  In weeks 1-8, 2 sessions per week were performed at an intensity of 70% 1-RM, where 3 sets of 10 repetitions were the last set was performed to momentary muscular failure.  Each session consisted of whole body resistance exercise that lasted ~60-70 min. To reduce the risk of injury and overtraining, one additional training session per week was conducted at 3 sets of 10 repetitions at 60% 1RM with the goal of not reaching momentary muscular failure.  These sessions took place immediately before and after the 1-RM training days.  In weeks 9-12, 2 sessions per week were performed at an intensity of 80% 1-RM, where 4 sets of 8 repetitions were performed to momentary muscular failure. The 3rd session was performed at an intensity of 60% 1-RM as indicated earlier.   Each session consisted of whole body resistance exercise that lasted ~70-90 min. Resistance exercises included flat and incline chest press; leg press, curl and extension; seated pull-downs and rows; calf raises; and abdominal exercises.  Participants rested for 1-2 minutes between exercises and individuals sets.  1-RM was directly tested on the chest press, leg press and knee extension" (Reidy 2016b).  

Participants were allowed to maintain their recreational physical activity but instructed not to do any other strength training outside of the study. To allow for unforeseen life events, participants were given 13 weeks following the familiarization period to complete 36 exercise sessions. This allowed for 100% exercise compliance.

Table 1: Summary of all protein supplement studies with a placebo group directly assessing muscle mass during RET in young adults (Reidy 2017).

Are DXA-measured lean mass gains in this and other studies misleading? As Reidy et al. point out in the discussion of the results, you'd need ~62 to ~140 participants "to find a statistical effect of protein supplementation on whole body lean mass or fat-free mass" - most of the existing trials don't have that many subjects (cf. Reidy 2016a,b). In addition, Reidy's very own study shows that despite an (almost) statistically significant increase in lean mass, an increase in local muscle hypertrophy, as measured by ultrasound in the legs, could not be established. In fact, the increase in lean mass the DEXA scan detected could have occurred everywhere and it's by no means certain that it took place in those areas of the upper body and arms where you want your gains to become visible.

On top of their habitually "high" protein intake (101-108g/day and 102-113g/day in the protein blend and whey group, respectively), all subjects were randomized to receive either one out of two daily 22g protein supplements containing ...

• a soy-dairy protein blend (PB, N=22) - 25% soy protein isolate, 25% whey protein isolate, and 50% sodium caseinate,2.00 g leucine from the three protein sources
• a whey protein isolate (WP, N=15) - 100% whey protein isolate, 2.31g leucine from whey 

... or an isocaloric maltodextrin and otherwise identical placebo (MDP, N=17) supplement. In view of the results, it may be worth noting that this left the subjects in the placebo group (MDP), who also had the lowest dietary protein intake (95g/day), with ~30g protein less those in the other groups.

Figure 3: While the scientists recorded sign. increases in muscle thickness in the subjects' vastus intermedius, these changes, however, were - within statistical margins - identical for all trials (Reidy 2016b).

Nevertheless, there were no significant differences in either of the main research outcomes, i.e. the lean mass (DXA data), vastus lateralis myofiber-type-specific cross-sectional-area, satellite cell content and myonuclear addition, which were assessed pre and post-resistance training. A closer analysis of the data reveals:

• 

  Table 2: Total energy and macronutrient intake during the study (from Reidy 2016b, which is the large clinical of which the study at hand is a subset)

  the soy-blend with its fast-to-slow digesting mix of 25% soy protein isolate, 25% whey protein isolate, and 50% sodium caseinate yielded the most sign. gains in lean body mass (p = 0.057 for PB), 
• adding protein, in general, i.e. pooling the results from PB and WP to PRO, increased the statistical sign. of the lean mass benefit to p = 0.050 (no sign. difference to MPB, still),
• despite the previously hinted at advantage of the blend, significant inter-group differences for soy-blend vs. whey were not observed for any of the parameters

Very similar observations had been made in the 2016(b) paper on this large-scale clinical trial, in which the scientists didn't find significant treatment effects (see TRT in Figure 4) of either of the protein supplements on DXA measures increases in lean body mass or lean arm mass.

Figure 4: Upon closer scrutiny, the lean mass data reveals a non-sign. advantage for the protein blend (Reidy 2016b).

Against that background, it will probably not surprise you that, again on a treatment basis (no pooling of the two protein groups), no differences were reported for the separately measured leg muscle hypertrophy and vastus lateralis myofiber-type-specific cross-sectional-area (P<0.05 | not shown in any figure) in the latest follow-up (Reidy 2017).

But what about the "more helps more" studies by Antonio et al.? I have to admit that I cannot fully explain why Jose Antonio and colleagues saw much more significant increases in muscle gains in their often-cited study. It could be that this is a methodological issue Reidy et al. address in their discussion of the results with the increases in total lean mass measured by DXA being sign. more pronounced than the actual changes in muscle cross-sectional area (compare box in the bottom line of this article), which were not measured in the 2014 study by Antonio et al. On the other hand, it is likewise possible that it's simply a question of the amount of protein that's supplemented. In the three-year-old study that was enough to bump the subject's protein intake to 4.4g/kg body weight. Anyway... Antonio's observations do not refute the conclusion that a single protein shake won't do the muscle-building magic you're promised on the labels of the bazillion of different protein products on the market.

Similarly, no treatment effects, i.e. effects due to a certain protein powder, were detected for the albeit highly significant training-induced changes in myosin heavy chain I and II myofiber satellite cell content and myonuclei content (P<0.05).

Figure 5: Change in the relative frequency of larger vastus lateralis MHC II myofibers. Protein blend (PB) or whey protein (WP) or maltodextrin placebo (MDP). Data are mean ± SEM. TRT, treatment (Reidy 2017).

Only when the scientists pooled the results of the two protein groups, they found a non-significant and very modest effect of protein supplementation on the increase in MHC I satellite cell content, isokinetic torque and a slight expansion of a greater proportion of larger MHC II fibers over placebo after resistance exercise training - a "benefit" that is nothing like those you'd expect if you read the grandiose promises in the shiny advertisements of the supplement industry.

Brainwashed by shiny ads, people tend to overesti-mate the benefits of protein supps: Reidy et al. make a valid point, when they write that "[c]ontrary to popu-lar dogma, it is not unusual to observe no effect of protein supplements, in particular, whey protein, over placebo on lean mass or myofiber CSA." The authors further point out that they "are aware of only 3 studies demonstra-ting greater changes in vastus lateralis myofiber CSA and 2 studies with magnetic resonance imaging (MRI) comparing protein versus carbohydrate placebo supp-lementation during RET" (Redy 2017). Similarly dis-appointing are the results of most studies that investiga-ted the vastus lateralis myofi-ber CSA in protein supple-mented subjects on protein-adequate diets. And that's no leg-specific result, as Reidy et al. highlight: "Studies utili-zing MRI of the biceps or la-tissimus dorsi and ultrasound of the thigh muscles have clearly shown the same pattern" (Reidy 2017). A simple measuring tape may thus indeed yield more relevant results than DEXA.

So what does all that tell me? Well, I guess the first thing you have to admit to yourself is that you're expecting whey... ah, I mean, way too much from your protein powder. As Reidy et al. point out "[p]rotein supplementation during resistance training has [only] a modest effect on whole body lean mass as compared to exercise training without protein supplementation" - especially if you have a relatively high (1.2g/kg or more) baseline protein intake.

Furthermore, the study, or rather the set of papers on this uniquely large clinical trial, should remind you of the potential benefits of protein blends. Why? Well, previous studies suggest that the combination of fast and slow proteins will provide for a more sustained and eventually more anabolic state of hyperamino-acidemia compared to fast-digesting proteins like whey protein isolate, alone. That this benefit was only small in the study at hand could be a mere result of the fact that a truly slow digesting source was missing from the protein blend (sodium caseinate contains no intact micelles and is digested much faster than micellar casein).

All that doesn't render protein supplements useless, but it emphasizes that protein is mostly the fuel for hypertrophy - not more, but also not less. Its provision can increase the storage of muscle protein but it will, as the large-scale clinical trial at hand has shown quite conclusively, not "enhance resistance exercise-induced increases in myofiber hypertrophy, satellite cell content or myonuclear addition in young healthy men" (Reidy 2017), significantly. Is that surprising? Well at least for the structural parameters it isn't as discussed in the "Intermittent Thoughts on Building Muscle" (see conclusion + article overview) the latter are not directly affected by mTOR and other signaling proteins an increased protein intake would affect. And if you still believed that the post-workout shake of yours would add another inch to your biceps every two weeks, you cannot be helped, anyway | Comment!

References:

• Antonio, Jose, et al. "The effects of consuming a high protein diet (4.4 g/kg/d) on body composition in resistance-trained individuals." Journal of the International Society of Sports Nutrition 11.1 (2014): 19.
• Reidy, Paul T., et al. "Protein supplementation has minimal effects on muscle adaptations during resistance exercise training in young men: A double-blind randomized clinical trial." The Journal of nutrition 146.9 (2016a): 1660-1669.
• Reidy, Paul T., and Blake B. Rasmussen. "Role of ingested amino acids and protein in the promotion of resistance exercise–induced muscle protein anabolism." The Journal of nutrition (2016b): jn203208.
• Reidy, Paul T., et al. "Protein Supplementation Does Not Affect Myogenic Adaptations to Resistance Training." Medicine & Science in Sports & Exercise (2017).

Study Probes Muscle Building Effects of Vitamin D in Young and Old and Finds None, but Relative Strength in Old and Fiber Composition & Myostatin in Young Muscle Respond

Old or young, who is going to benefit and who is going to benefit most from vitamin D supplementation during a 12-week resistance training regimen. Unfortunately, we don't have an unambiguous answer - yet!?

Ok, I have to admit, I could have kept up the suspense by not giving away the main result of Jakob Agergaard's and colleagues' latest study in the headline, already. On the other hand, by giving away the most relevant information in the headline, I can make sure that future google searchers will immediately refute the claim that "vitamin D is a powerful muscle builder" - it is not. What it may very well be, is a vitamin that is necessary for your long-term success.

This is still much different from what you may conclude solely based on the associations that exist between low vitamin D and all sorts of ailments, though. Evidence that vitamin D(3) supplements are able to reduce the risk of bone fractures, diabetes, cardiovascular diseases, cancer, depression, osteoarthritis, multiple sclerosis, and other immune-related diseases is still preliminary. Very unfortunate in view of big research dollars that have been spent without yielding D-finite results and hundreds of more or less practically useless observational studies.

There are many ways to get your vitamin D - learn more the SuppVersity

How Much Vit D Do I Take?

Losin' Fat'll Raise D; Not Vice-Versa

Vit. D Speeds Up Recovery

Overlooked D-Sources

Vitamin D For Athletes!

Vitamin D Helps Store Fat

I was thus very happy to see that the scientists from the University of Copenhagen did not content themselves with correlating the individual gains of their young and old subjects with the corresponding vitamin D levels. Instead, they designed a randomized controlled trial in which they "investigate[d] whether vitamin-D intake during 12 weeks of resistance training has an additive effect on muscle hypertrophy and strength"  (Agergaard. 2015) in Healthy, sedentary young (aged 20–30 years) and elderly (aged 60–75 years) Caucasian men living within the local community in Copenhagen:

"We hypothesized that intake of vitamin-D plus calcium would improve the outcome of three months of resistance training in healthy untrained individuals resulting in greater muscle strength and hypertrophy compared to a training control-group supplemented with calcium alone (placebo). Moreover, we hypothesized that resistance exercise would increase the mRNA expression of VDR and CYP27B1. The study included a group of young and a group of elderly individuals to elucidate a possible blunted hypertrophic response in the aging muscle"  (Agergaard. 2015).

The study took place at Bispebjerg Hospital, Copenhagen, Denmark (latitude of 56°N). Inclusion was continuous from November 17, 2010, to December 21, 2010, and the last subject completed the study on April 25, 2011. Thus, the study was conducted in a period of low UVB irradiation from sunlight. The risk of interference by uncontrolled sun-exposure was thus low. About as low as I suppose some of you will say the supplementation dose was. The latter consisted of either

• placebo supplementation with 800mg of calcium per day, or
• vitamin D + calcium at a dosage of 48µg (1920 IU) vitamin-D 3 + 800 mg calcium/day

that was administered in two servings, with one tablet containing 10 μg vitamin-D 3 + 400 mg calcium and one tablet containing 38 μg vitamin-D 3 + 400 mg calcium and had to be taken with meals (this increases absorption | learn more).

You're too lazy to read and want some extra-information, also on the topic of fat cell cellularity, obesity and body weight regain (yo-yo effect?) - Download yesterday's installment of Super Human Radio and listen to my interview an add-free version right here!

The scientists probably would have dosed higher, but since the maximum advisable daily dose according to the Danish Health and Medicines Authority is 50 μg, i.e. 2000 IU, they probably felt that their hands were tied.

Figure 1: Flowchart showing a young and b elderly subjects from first contact to end of study (Agergaard. 2015)

All subjects who had been randomly (double-blind) assigned to the respective group had to follow the same standardized workout routine consisting of a total of 36 training sessions (12 weeks with 3 sessions/week) with 5–10 min warm-up on cycle ergometers followed by resistance training exercises of the lower extremities (only!) performed in commercial knee extension and leg press devices (Technogym, Super Executive Line, Gambettola, Italy) in each session. All sessions were supervised. Progressive loading levels were monitored continuously and adjusted throughout the entire training period to maintain muscle loading at the intended values.

• During the first 6 training sessions, participants completed 3 sets of 12–15 repetitions at 65–70 % of 1RM. 
• During session 7–12, participants performed 3 sets of 10–12 repetitions at 70–75 % of 1RM, increasing to 4 sets at 70–75 % of 1RM during session 13–18. 
• From session 19 and onwards, participants performed 5 sets with training load progressing from 8–10 repetitions at 75–80 % of 1RM in session 19–27 to 6–8 repetitions at 80–85 % of 1RM in session 28–36 [38]. 

The exercises were performed in a moderately slow, controlled manner with 1–2 s in the concentric- and eccentric phase with a rest of 1–3 min between sets. Exercise compliance (sets, repetitions, and load) was calculated from daily exercise records completed by the instructors at each training session. Participants were informed that a mean attendance of less than 2 training sessions per week resulted in exclusion. All adverse events associated with the training intervention were recorded.

The complex ways in which vitamin D supplements interact with both the levels of the active form of vitamin D 1,25(OH)2D and the binding proteins vitamin D binding protein and serum album has yet not been considered in any of the "vitamin D and gains" studies - epic fail ? (data from Glendenning. 2015)

Vitamin D Binding Protein, Bioavailable Vitamin D & Receptor Polymorphisms - Although it has been known for decades that only 0.1% of the vitamin D in our body and only ~10% of the metabolites in our blood are free, the effects of being bound to its specific binding protein (VDBP) or albumin are still largely unknown. One of the reasons is that studies still rely on unreliable measurements of total vitamin D that are then run through algorithms to elucidate if there's a difference between the effects of free and bound vitamin D (Chun. 2014). This is not only problematic because it assumes that we'd all have the same / similar amounts of vitamin D binding protein, but also because it ignores already established genetic polymorphisms (e.g. inter-racial / whites are more likely to have a low binding affinity than blacks) in how VDBP works and how it affects our health and is affected by supplementing with vitamin D (sign. increases are seen w/ vit D2 or D3 | see Fig.).

A similar negligence can be observed with regard to the role of the vitamin D receptors on its various target organs. While we know that their expression increases with resistance training (no added increase was observed with vitamin D supplementation in the study at hand in contrast to a recent study by Makanae et al. (2015) in rodents), we still have almost no clue how they interact with free and bound vitamin D; and only recently researchers like Jia et al. (2015) have begun to investigate how certain vitamin D receptor polymorphisms (gene types) like the rs739837 gene are associated with increased risk of T2DM. In conjunction with the role of genetic polymorphisms of the binding proteins, the whole system is at the moment, thus way too too complex for us to make predictions on a population or even sub-population levels (like the elderly, men and women at an increased risk of cancer, or patients with autoimmune diseases, or athletes). 

The outcome variables the scientists choose were skeletal muscle hypertrophy, isometric muscle strength, serum vitamin D levels, and a muscle biopsy that was used (a) to analyze several markers of muscle hypertrophy, metabolism & co, as well as (b) to determine whether training or treatment had triggered measurable or even significant changes in the fiber type composition of the subjects.

Figure 2: Serum vitamin D levels at all time-points during the study (I added the markups for the zones to the original figure from Agergaard to make it easier for you to interpret the data).

Of these, I deliberately chose the 25OHD serum levels to start with. Why? Well if you look at the small increase in the young subjects and the still existing gap between their 25OHD (=serum vitamin D) levels and the allegedly "optimal" zone for lower body strength gains (cf. Bischoff-Ferrari. 2006), you may feel reassured that the dosages were too low. This is yet only the case, if the goal was to get the levels into the "magic" 90-100nmol/L of which Bischoff-Ferrari estimated in 2006 that it was optimal for muscle function and health. Whether the effects would have been more pronounced if the subjects had reached this level is yet mere speculation and, if you look at the correlation analysis further down, even highly unlikely (see Figure 5 and respective explanations).

Figure 3: Cross sectional area (CSA), Isometric strength and strength/CSA of Quadriceps muscle. Change in a CSA, b isometric strength and c strength/CSA of quadriceps muscle for young and elderly vitamin-D and placebo groups, respectively. Data shown as mean percentage change from week 0 ± SEM. * different from week 0 (p < 0.05)

Now, as arbitrary as these ranges may be (things like the influence of the vitamin D binding protein levels and genotypes for example, are taken into account, at all | Chun. 2014; Koplin. 2015), we must not and will not ignore the fact that the young, unlike the old(er) subjects, didn't make it into 90-100 nmol/L zone of "magic gains" when we are looking at the data in Figure 3:

• No group effects - The first thing you should realize is that there were no significant inter-group differences and thus no group effects in response to the provision of vitamin D3 vs. placebo. This does imply that neither the increased size gains (A) in the vitamin D group in the young nor the decreased gains in the vitamin D group in the old subjects was statistically significant. The same can be said, albeit in the opposite directions for the strength increases (B) and the relative strength increases (C) in the young subjects.
• Significant time effects - Since subjects in both groups still gained significant amounts of muscle and strength, the one thing the study does confirm is the efficacy of resistance training as strength and mass builder in young and old.
• Significant group effect on relative strength in the elderly - Due to the reversal of the observations compared to the young group (lower size gains + higher strength gains in the older, higher size + lower strength gains in the younger subjects), the relative strength of the older subjects has improved by vitamin D supplementation (p = 0.008, not correctly indicated in Figure 3) - a result that stands in line with previous research like Moreira-Pfrimer et al. (2009) where the provision of 150,000 IU once a month during the first 2 months, followed by 90,000 IU once a month for another 4 months enhanced both, the 25(OH)D levels and the lower limb muscle strength of the > or =60 year old subjects, even in the absence of any regular physical exercise practice.

Now, I would be inclined to ignore the lack of statistical significance for the initially mentioned parameters and jump on the significant increase in the older subjects and the trends we may extrapolate from the rest of the data if it were not for the results of the extra correlation analysis the scientists did. If higher levels of vitamin D3 (90-100nmol/L as they were achieved in the older subjects) could, as Bischoff-Ferrari et al. assume based on observations Guralnik, et al. (1995) and Seeman et al. made in elderly individuals, ameliorate exercise-induced strength gains in the young subjects, there should at least be a correlation between vitamin 25OHD levels and muscle size and strength similar to the one Bischoff-Ferrari et al. report for the 8-foot-walk and sit-to-stand test:

Figure 4: The optimal ranges, Bischoff-Ferrari et al. estimated are based on the above observational data from a 8-foot-walk and sit-to-stand test done in the elderly. That's super reliable and just like you, right? No? Well, that's why I believe those "optimal values" have no relevance for the young and low relevance for the old subjects (Bischoff-Ferrari. 2006)

If the trends you may believe to see in Figure 3 a-c remained trends, because the 25OHD levels didn't rise high enough, the graphs in Figure 5 would look much different: They would firstly show increasing, not no or decreasing slopes and would second of all provide evidence for a practically relevant correlation between the 25OHD levels, the muscle size, strength and relative strength.

Figure 5: Correlation between Quadriceps ΔCSA, ΔIsometric strength, Δstrength/CSA and 25(OH)D (Agergaard. 2015)

In practice, however, the correlation analysis yielded nothing: No correlation between 25OHD and size gains (A), no correlation between 25OHD and strength gains (B), and no correlation between 25OHD and relative strength gains (C). While this does not neglect the possibility that the vitamin D supplement still affected the increase in strength/size ratio of the elderly, the result warrants the conclusion that there was "[n]o additive effect of vitamin-D intake during 12 weeks of resistance training [...] on either whole muscle hypertrophy or muscle strength" (Agergaard. 2015).

So vitamin D supplementation is finally disproven? It is not just the specific study population (unhealthy individuals or athletes may benefit more, men and women may differ (Ko. 2015) etc.) that precludes making overgeneralized conclusions such as "vitamin D supplementation doesn't do anything for your gains". There is more! Firstly, there is the increase in what the scientists call "muscle quality", i.e. the ratio of strength/size increases in the elderly. Now, the data in Figure 5 indicates that this is clearly not a function of the serum 25OHD levels. If that's not the case, however, it could only be mediated by vitamin D3 directly or metabolites that haven't been tested in the study at hand (most prominently active vitamin D, i.e. 1,25-dihydroxycholecalciferol aka calcitriol). If that's the case, age may explain that the older subjects did not see the same changes in fiber type morphology (greater increase in type IIa) and myostatin expression the young ones did.

Figure 6: Significant treatment specific changes in fiber type (%), i.e. increases in fast-twitch type IIa fibers and decreases of the protein synthesis inhibitor myostatin were observed only in younger subjects (Agergaard. 2015).

I highlighted these changes with arrows in Figure 6 and would like to point out that they are the most interesting reason to still supplement w/ vitamin D. Eventually, both effects could affect your gains in the long-term: (I) lower myostatin = higher protein synthesis; (II) more type IIa fibers = higher growth potential. In only 12-weeks, however, newbies don't reach a level where myostatin and/or the fiber composition of their muscle is holding them back, significantly. For athlete and after longer training periods, however, the scientifically proven (albeit in vitro | Garcia. 2013, 2014)  ability of active vitamin D aka calcitriol (and / or vitamin D3 directly - not proven in human muscle) to increase the myogenic differentiation (would explain myofiber changes) and suppress myostatin in human myoblasts could turn out to be game changers.

To find out whether these purported long-term effects exist and/or if similar effects can be seen in non-sedentary adults, like athletes who would benefit the most of reduced myostatin levels and further changes in the muscle architecture, we do yet need more studies. Randomized controlled studies, maybe with different dosing schemes (the ~2,000 IU are not exactly much if we consider potential direct effects) and no more observational bogus on vitamin D | Comment on Facebook!

References:

• Agergaard, Jakob, et al. "Does vitamin-D intake during resistance training improve the skeletal muscle hypertrophic and strength response in young and elderly men?–a randomized controlled trial." Nutrition & metabolism 12.1 (2015): 32.
• Bischoff-Ferrari, Heike A., et al. "Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes." The American journal of clinical nutrition 84.1 (2006): 18-28.
• Chun, Rene F., et al. "Vitamin D and DBP: the free hormone hypothesis revisited." The Journal of steroid biochemistry and molecular biology 144 (2014): 132-137.
• Garcia, Leah A., et al. "1, 25 (OH) 2 vitamin D 3 enhances myogenic differentiation by modulating the expression of key angiogenic growth factors and angiogenic inhibitors in C 2 C 12 skeletal muscle cells." The Journal of steroid biochemistry and molecular biology 133 (2013): 1-11.
• Garcia, Leah A., et al. "1, 25 (OH) 2vitamin D3 stimulates myogenic differentiation by inhibiting cell proliferation and modulating the expression of promyogenic growth factors and myostatin in C2C12 skeletal muscle cells." Endocrinology 152.8 (2011): 2976-2986.
• Glendenning, Paul, et al. "Calculated free and bioavailable vitamin D metabolite concentrations in vitamin D-deficient hip fracture patients after supplementation with cholecalciferol and ergocalciferol." Bone 56.2 (2013): 271-275.
• Guralnik, Jack M., et al. "Lower-extremity function in persons over the age of 70 years as a predictor of subsequent disability." New England Journal of Medicine 332.9 (1995): 556-562.
• Jia et al. "Vitamin D Receptor Genetic Polymorphism Is Significantly Associated with Risk of Type 2 Diabetes Mellitus in Chinese Han Population." Arch Med Res. (2015): Ahead of print. 
• Ko, Min Jung, et al. "Relation of serum 25-hydroxyvitamin D status with skeletal muscle mass by sex and age group among Korean adults." British Journal of Nutrition (2015): 1-7.
• Koplin, Jennifer J., et al. "Polymorphisms affecting vitamin D–binding protein modify the relationship between serum vitamin D (25 [OH] D 3) and food allergy." Journal of Allergy and Clinical Immunology (2015).
• Makanae, Yuhei, et al. "Acute bout of resistance exercise increases vitamin D receptor protein expression in rat skeletal muscle." Experimental physiology 100.10 (2015): 1168-1176.
• Moreira-Pfrimer, Linda DF, et al. "Treatment of vitamin D deficiency increases lower limb muscle strength in institutionalized older people independently of regular physical activity: a randomized double-blind controlled trial." Annals of Nutrition and Metabolism 54.4 (2009): 291-300.

The Way You Train Shapes Your Muscle Size and Function - Study in Powerlifters, Bodybuilders & Controls Suggests Effects Go Beyond Hypertrophy & MHC Fiber Composition

Don't worry, this exercise is not going to reverse all the effects of your training. Still, in view of the intriguing results of the study at hand, it would be interesting if the fiber-type unspecific effects in powerlifters are reversed when you stop powerlifting / exercise altogether and/or start lifting with higher volumes and lower intensities (bodybuilding style volume training).

There are visible and invisible differences between bodybuilders, powerlifters and normal men. The former ones are so obvious that it wouldn't be very interesting to address them in a study. The latter, on the other hand, are, other than you'd expect, hardly researched, but - as the results of a recent study from the Manchester Metropolitan University, and a bunch of other European Universities and research centers shows - more pronounced and significantly more fundamental than some of you may have thought.

As Meijer et al. point out in the introduction to their soon-to-be-published paper in the peer-reviewed scientific journal Experimental Physiology, the performance of a power athlete is largely determined by two traits: the maximal force and power generating capacity of the recruited muscles, and the ability to maintain force and power for a prolonged period of high intensity efforts.

Want to get stronger, bigger, faster and leaner? Periodize appropriately!

30% More on the Big Three: Squat, DL, BP!

Block Periodization Done Right

Linear vs. Undulating Periodizationt

12% Body Fat in 12 Weeks W/ Periodizatoin

Detraining + Periodization - How to?

Tapering 101 - Learn How It's Done!

Needless to say that the peak muscle power and force will to some extent dependent on muscle volume and physiological cross-sectional area of the muscle, respectively. In accordance with the above definition of what one's power will depend on, though, the mitochondrial density in the recruited muscle fibres does yet figure as well. I mean, you don't want to run out of steam on your 3rd rep of squats, do you? That's what I thought. Thus, Meijer et al. are right to point out that "[i]deally, an  athlete seeks to maximize both muscle power and endurance" (Meijer. 2015).

Myostatin Limits Muscle Hypertrophy in Everyone! Even Normal Gymrats May Benefit from Blockers, if any of them Actually Worked  Learn more!

The problem with this maxim, however, is that you cannot maximize both, the fibre cross-sectional area (FCSA) and its mitochondrial density. In fact, studies suggest that the FCSA at a given mitochondrial density is limited by the maximal extracellular oxygen tension (Van der Laarse et al., 1997; Wessel et al., 2010) or the myonuclear domain sizes (learn more).

It is thus only logical that studies on myostatin negative animals show that limit the amount of hypertrophy beyond which it becomes disadvantageous for sustainable power (learn more). Weak big, vs. small(er), yet strong muscles. That sounds like powerlifting vs. bodybuilding, right?

Although the fiber composition of bodybuilders, gymrats, endurance rowers and sedentary control differs, it is not - according to the results of the study at hand - the only and maybe not even an important determinant of maximal power (data based on Jürimäe. 1997)

Hold on, why's that even newsworthy? As the authors themselves highlight, their study is the first to show that "long-term resistance exercise, represented here by power athletes and body builders, increases the force generating capacity of muscle fibres" (Meijer. 2015). Now, this alone is not really exciting, what is intriguing, though, is that "this increase in force was only in power athletes associated with a significant increase in the power generating capacity of single muscle fibres" (ibid.) - and thus largely independent of the fiber type composition of the muscles (learn more).

Accordingly, on single fiber basis, the power generating capacity of a body builder (BB) is not only lower than that of a powerlifter, it's actually not even higher than that of an untrained individual (C) - and that despite the fact that the individual muscle fibers are significantly larger. As the authors further explain, "[t]his unexpected observation was explicable by a lower fiber specific tension in BB compared to C fibres". In this context it is important to point out that the C and PA group in the study at hand performed a comparable, albeit relatively low amount of aerobic exercise, it is thus "likely that the effects shown in PA can be attributed to the high-intensity low-volume resistance training," (ibid.), alone.

Well, that's obviously what Meijer et al. thought, as well, when they phrased the following hypotheses about the way the muscle fiber specific tension (F0) of 12 male bodybuilders (BB | BB: 29.8 ± 4.8y; 177.8 ± 4.1 cm; 91.7 ± 13.4 kg), 6 male powerlifters / power athlete (PA | 23.4 ± 3.9 y; 185.0 ± 4.3 cm; 103.0 ± 7.3 kg) and 14 non-competitive / weight lifting controls (C | 24.0 ± 3.5 y; 180.9 ± 5.3 cm; 77.9 ± 6.3 kg) differ in the introduction to their latest paper:

"PAs is characterized by high-intensity low-volume resistance training with supplemental aerobic exercise. Since BBs train for bulk and PAs for function we hypothesize that fibres from PAs will have a higher specific power and F0 than those from BBs. We expect an increase in specific power and F0 in PAs and BBs (for BBs especially in type II fibres) compared to C." (Meijer. 2015). 

To test whether this hypothesis is correct, the researchers compared muscle fibre contractile properties of biopsies taken from m. vastus lateralis of 12 bodybuilders (BB; low- to moderate-intensity high-volume resistance training), 6 power athletes (PA; high-intensity low-volume combined with aerobic training) and 14 controls (C). To do that you have to take samples from the muscle and test the maximal isotonic contractions on a single muscle fibre in vitro.

Figure 1: Typical examples of three IIA muscle fibres. Circles represent the measured force-velocity data. The continuous line shows the fitted force-velocity curve and the dashed line shows force-power curve (top). Specific tension of skinned m. vastus lateralis fibres is inversely related to muscle fibre cross-sectional area (bottom) - For both rows of data: Non-resistance trained controls (left), bodybuilders (middle), power athletes (right | Meijer. 2015).

As the data in Figure 1 shows, the scientists' analysis of their subjects' muscle fibers produced unsurprising and surprising results:

• BBS have larger muscle fibers (unsurprising) - The fibre cross-sectional area (FCSA) was 67% and 88% (P<0.01) larger in BB than in PA and C, respectively.
• There's no difference in fiber size between C and PA (surprising?) - Unlike the difference between bodybuilders, power athletes and control, the existing difference in fiber size between PA and C did not reach statistical significance. 
• BB and PA fibres are stronger (unsurprsing) - BB and PA fibers developed a higher maximal isometric tensions (32%, 50%, P < 0.01) than those of C. 
• BB & C fibers are significantly weaker than PA fibers (unsurprising) - The specific tension (F0) of BB fibres was 62% and 41% lower than that of PA and C fibres (P < 0.05), respectively. 
• The increased peak power of PA fibres was not related to fibre type (surprising) - Irrespective of fibre type, peak power (P) of PA fibres was 58% higher than that of BB fibres (P < 0.05), while BB fibres –despite considerable hypertrophy- had similar PP as C fibres. 

With the latter result standing in contrast to the long-held believe that training induced changes in fiber composition like an increase in the proportion of power-specific MHC IIa,b fibers in power athletes and an increase in "enduring" type II fibers (MHC IIx) in bodybuilders were responsible for the strength and power differences, the study at hand provides initial evidence that that the effects of the way you train go way beyond selective type IIa,b vs. type IIx hypertrophy.

Sedentary Individuals, Endurance & Strength Athletes: Their Fitness, Training & Hormones and How They Effect the Ratio of Fast- to Slow-Twitch Fibers | learn more.

Bottom line: The study at hand provides the first reliable evidence that high-intensity low-volume resistance training as it is performed by power lifters triggers significantly different adaptational processes than the low- to moderate-intensity high-volume resistance of bodybuilders - adaptational processes that go beyond the fiber-specific hypertrophy effects that are responsible for the alleged muscle fiber composition changes in trainees.

Since the former is a completely novel result, we can only speculate about the underlying mechanism. Meijer et al. "postulate that the decrease in specific tension is caused by differences in myofibrillar density and/or post-translational modifications of contractile proteins" (Meijer. 2015).

That's obviously a very unspecific hypothesis that warrants further investigation and elaboration in future studies; not just to confirm it, but also to elucidate (a) the time it takes for these changes to take place and (b) whether they are reversible by (1) changing the way you train (2) staying away from the gym, altogether | Comment on Facebook!

References:

• Jürimäe, Jaak, et al. "Differences in muscle contractile characteristics among bodybuilders, endurance trainers and control subjects." European journal of applied physiology and occupational physiology 75.4 (1997): 357-362.
• Meijer et al. "Single muscle fibre contractile properties differ between bodybuilders, power athletes and controls." Experimental Physiology (2015): Accepted article.
• Van Der Laarse, W. J., et al. "Size principle of striated muscle cells." Netherlands journal of zoology 48.3 (1997): 213-223.
• Van Wessel, T., et al. "The muscle fiber type–fiber size paradox: hypertrophy or oxidative metabolism?." European journal of applied physiology 110.4 (2010): 665-694.

Sedentary Individuals, Endurance & Strength Athletes: Their Fitness, Training & Hormones and How They Effect the Ratio of Fast- to Slow-Twitch Fibers

ATPase stained cross-section (100x magnification) of the quadriceps muscle of an untrained adult. Type 1 fibers are light, type 2 fibers are dark (McDonald. 2002). Can you change the make up? Is it genetically predetermined? Or is it determine by your hormones - and if so, which ones are responsible - "The Big T", again? Read more about testosterone's role in building muscle, here!

Wednesday, last week, I discussed the immediate effects of "cardio before strength training" vs. "cardio after strength training" and came to the conclusion that the prolonged yet acute elevation of "anabolism" (as measured by higher testosterone levels) in the "cardio before" group is of little significance with respect to the real-world effects the concomitant training regimen is going to have on the hypertrophy and strength gains of the average trainee.

Why I am telling you all that? Well, actually I wanted to post this news item last Thursday already, but then Adelfo's contest prep update appeared out of nowhere and I had to postpone the study on the "skeletal muscle myosin heavy chain isoform content in relation to gonadal hormones and anabolic-catabolic balance in trained and untrained men" I actually wanted to discuss as a follow-up to the Cardore study (cf. "Cardio First if You Want to Leave the Gym More "Anabolic" Than You Were When You Came in?").

Fast or slow muscles? Are your hormones to blame?

Contrary to the Brazilian researchers who put their study participants through two identical workouts, the scientists from the University School of Physical Education, the Jagiellonian University and the Cancer Institute in Krakow, Poland did an ex-post analysis of the influence of endurance and strength training on the gonadal hormones and anabolic-catabolic hormone balance as well as their influence on myosin heavy chain (MHC) transformation in humans and found that...

"[...] despite considerable dissimilarity in MHC content between endurance trained, sprint / strength-trained, and untrained men, there are no differences between them in regard to basal T concentration."(Grandys. 2012)

This may be in conflict with the bro-scientific notion that you just have to take your overpriced T-boosters (or better real gear) religiously and will convert all your (falsely) unloved type 1 into type 2 fibers, but is consistent with previous studies which compared the total testosterone levels of top-class sprinters with untrained subjects and were likewise unable to proof statistically significant difference between the two (Grandys. 2011).

Fit = more testosterone? No! If anything, fat and unfit = less testosterone

Moreover, the results clearly show that contrary to being fat and metabolically deranged, not working out alone does not have negative effects on your total testosterone levels (much contrary to overtraining, by the way). Now, this does certainly put another huge question-mark behind the "exercise induced increases in testosterone build muscle hypothesis". It stands to reason that the sprinters, were, just like the strength athletes in the study at hand more muscular than the sedentary men and had an increased amount of the hypertrophy prone MHC-II muscles (fast twitch, predominantly intermediate type), but if you look at the actual study outcome, you will have to concede that this appears to be a function of the training stimulus and the accommodation to either strength or endurance workouts and has little to nothing to do with the testosterone levels.

Figure 1: Conditioning parameters (PO2_max and VO2_peak; left) and MHC-fiber composition (right) of untrained (U), endurance trained (E) and strength / sprint (S) athletes (Grandys. 2012)

In fact, there were highly significant correlations between the fiber type distribution between the endurance-trained athletes (E), and sprint/strength-trained athletes (S) in the study at hand. For example, the scientists observed...

• statistically significant correlations between muscle fiber type and VO2_peak (r = -0.49, p < 0.01) , expressed both in absolute and relative terms, and power output observed at the VO2peak (POmax)  between the untrained (U; rel. VO2Peak 46ml/kg/min, p < 0.001), sprint training / strength training (S; rel. VO2Peak 47ml/kg/min, p=0.02) and endurance training (E; 55.9 ml/kg/min)
• even higher correlations between fiber type and PO2_max (r = -0.69, p < 0.01), which is the maximal power production when cycling at the VO2_peak

Now, while all that clearly indicates that what you do at the gym is the fundamental determinant of your MHC-fiber composition and with it at least to a certain degree your growth propensity (and obviously overall cardiovascular fitness), there is one thing that brings the hormonal response back to the forefront:

There is a statistically significant correlation (r = 0.69) between the MHC-II content of the muscle fibers and the free testosterone to cortisol ratio. 

Figure 2: Ratio of free testosterone to cortisol and MHC-2 content of the muscles. Given the fact that the fT levels did not show significant differences, the determining factor thus appears to be - once again - cortisol not testosterone.

With the baseline free testosterone levels being identical (~20nmol/L) and in the upper third of the normal range  (9-38nmol/L) the confounding variable here is yet - once again (!) - not testosterone but cortisol. In this case not as positive regulator of total increases in lean mass (cf. figure 2 in Wednessday's post), but as a modulator of skeletal muscle myosin heavy chain isoform composition, which is - and I would like to emphasis this - not a matter of losing one for the other, but rather a matter of expressing more of type A while keeping the total amount of the other relatively steady.  Please take another look at figure 1 in the "Intermittent Thoughts on Building Muscle" and take a mental note of the fact that bodybuilders have higher total MHC-1 fiber contents than endurance rowers and a higher ratio of MHC II (X) fibers to MHC I (II/I_ratio = 1.76) than rowers (II/I_ratio = 1.69).

So, what's the role of cortisol in this case and what are the implication for muscle-heads and endurance junkies?

With cortisol being increased in response to both extreme high volume and endurance training (long and "in the fat burning zone", which is basically also the "zone" in which you would be running a non-competitive 10k if you are well-trained), you better limit both these training types to a reasonable amount if you want to maximize your MHC-II fiber count and more importantly, don't want to overtrain.

That it's neither necessary nor feasible to totally neglect one type of training for the other should yet be obvious by (a) the necessity to keep some muscle as "metabolic currency" and life-insurance, for the time the average and extraordinary endurance athlete gets older, and (b) the fiber composition of bodybuilders, which is not half as MHC-II dominant as bro-science would have it. And even if you "only" want to get stronger - do you really want to have to drop the weight because you are running out of steam at the bottom of a 400lbs squat? I don't think so...

References:

• Grandys M, Majerczak J, Zapart-Bukowska J, Kulpa J, Zoladz JA. Gonadal hormone status in highly trained sprinters and in untrained men. J Strength Cond Res. 2011 Apr;25(4):1079-84.
• Grandys M, Majerczak J, Karasinski J, Kulpa J, Zoladz JA. Skeletal muscle Myosin heavy chain isoform content in relation to gonadal hormones and anabolic-catabolic balance in trained and untrained men. J Strength Cond Res. 2012 Dec;26(12):3262-9.
• McDonald W. Basic Histochemistry and Electron Microscopy of Normal Muscle. University of South Florida. 2002 < http://missinglink.ucsf.edu/lm/ids_104_musclenerve_path/student_musclenerve/normal2.html > retrieved on  01/06/2013

Speed Up Your Regeneration and Propel Your Gains by Taking a HOT Bath Bath 2-Days Before Arduous Workouts

Image 1: Are women tougher than men, because bathe more often? If you define toughness by your muscles resistance to eccentric exercise damage, the answer could be "YES!"

If you listened to Brooks, Carl and me on Super Human Radio, yesterday (download the podcast), you may remember me stating that 48h appears to be a good rule of thumb, as far as the rest periods between workouts for individual body parts are concerned (this assumes that you are young, healthy, reasonably conditioned and lift heavy). A recently published paper by Chad D. Touchberry  does now suggest that there may be another 48h window before your workout (Touchberry. 2012). One you would use a priori to improve your recovery a posteriori - preconditioning in a hot bath for 20 min at 41°C, 48h before a hard workout or competition!

Eccentric treadmill running = maximum muscle damage

At least in a rodent model, those 20 min of heat exposure in 41°C warm water lead to statistically highly significant decreases in exercise induced muscle damage, improved and accelerated the recovery process and, contrary to what could be assumed based on previous research on the expression of heat proteins (Frier. 2007), did not hamper, but promote muscle gains in response to an exercise protocol consisting that consisted of running at 18m/min down a -16% grade for 5 min. This protocol has been used as a model for injurious exercise repeatedly in the past and constitutes one of the standard tests in rodent, but also in human studies (e.g. Pumpa. 2011).

Figure 1: Creatine kinase (CK) activity and immune cell infiltration after eccentric exercise with (EE+HS) and without (EE) preconditioning via hot bath 48h before (data calculated based on Touchberry. 2012)

As the data in figure 1 goes to show, the hot bath (EE+HS) had significant ameliorative effects on both the muscle damage (indicated by CK and the black sections in the H&E-stained soleus muscle cross-sections in figure 1, right), of which the researchers state that, despite the fact that "the mechanism by which heat shock protects skeletal muscle from damage is currently unknown", the protection of skeletal muscle against damage in mice overexpresssing HSP70 (McArdle. 2004a) as well as the differential HSP72 elevation in the HS group 2h and 48 h following exercise collectively

[...] suggest that HSP72 or another heat sensitive protein (i.e.,alphaB-crystallin) may play a role in mediating cytoprotection of skeletal muscle cells.

Moreover, Touchberry et al. explain the existing discrepancies between their own results and previous results by Mc.Ardle et al. (Mc. Ardle. 2004b), who did not find reduced muscle damage after pre-treatment with hsp-inducing concentric exercise 10h prior to the (in my humble opinion questionable) in-vitro application of eccentric strain to skeletal muscle tissue, with the "greater time for HSP accumulation prior to the exercise stressor" in their (48h) vs. the Mc.Ardle study (10h), which is obviously yet another indicator that rest is one of the most under-appreciated determiners of workout efficiency (cf. my words on SHR ;-)

Regeneration is one thing, but are muscle gains another?

Now, I am well aware that one of the main reasons regeneration isn't sexy, is that it does not trigger the phosphorylation of Akt, m-TOR and all the rest of the sciency terms with which laymen are bombarded by the supplement industry these days.

Figure 2: Total protein, new myosin heavy chain (MHCNEO) content and p-Akt expression in soleus muscle 2h and 48h after the eccentric exercise bout (data calculated based on Touchberry. 2012)

The study at hand does even show that heat pre-treatment will actually reduce, not promote the phosphorylation of AKT 48h after the exercise bout (cf. figure 2). If you do yet take into account that the total protein concentration and MHCneo (novel myosin-heavy-chain motor proteins) content in the soleus muscle of the rodents was increased profoundly, I guess you will have to agree that it is unlikely that less damage, a faster regeneration, and as a consequence less need for protein to be recruited via p-AKT only to repair the damage is going to propel, not diminish your gains!

Practical implications & open questions

Once again, the obvious message of this study is: Not he who trains the most, but he who regenerates and rebuilds the best, gains the most! And adequate rest aside, preconditioning in a hot (not a "cold thermogenic" bath ;-) can help dampen the exercise induced damage and accelerate your recovery.

Note: In February 2012, Bayley et al. published a paper that shows that the application of passive heat in form of a 42°C hot water bath for 40min immediately prior to a bout of HIT leg extensor exercises reduced the time to fatigue in seven healthy men by a whopping -36%  (Bayley. 2012). Impatience, or rather the unwillingness to grant your body the time it needs to recover is thus detrimental even if the stressor is "just" a hot bath!

Whether the same would be true if you train today and do the hot water immersion immediately, 2h, 10h or 12h post and thus 48h, 46h, 38h or 36h before your next workout is yet about as questionable, as whether or not similar effects could be elicited by switching back and forth between light and heavy days every 48h.  Both may appear likely, but aside form the fact that the optimal timing or workout intensity will still have to be elucidated, are still in the state of an interesting research hypothesis, not more, but also not less.

Update - Suggested reading: Since there have been questions pertaining to the usefulness of hydrotherapy post-workout, i.e. as a means of "classic" re- and not "precovery", I thought I rather refer you directly to my buddy Sean's E-book on the issue. Here is a snippet from the book

Image 2: Don't miss Sean's free e-book on classic hydrotherapy

Quick Hit Summary Water therapy is a common modality to enhance muscle recovery post workout. Sitting in chest high thermoneutral water for 20-30 minutes may accelerate waste removal while increasing blood flow to working muscles. Cold, hot and contrast water temps are also commonly used to assist recovery. The goal of cold water therapy is to reduce inflammation whereas hot water purportedly increases muscle blood flow. Contrast water therapy involves alternating between hot and cold water baths to induce a vaso-pumping effect. Current evidence does not support the theory behind these latter 2 therapies simply because the heat (from the water) is incapable of penetrating more than a couple centimeters into the skin. Thus, there is no stimulus to increase muscle blood flow

You can get this e-book alongside two other books for free if you register for Sean's newsletter, which is, take my word on it (!), not a weekly advertisement piece!
 
References:

1. Bailey SJ, Wilkerson DP, Fulford J, Jones AM. Influence of passive lower-body heating on muscle metabolic perturbation and high-intensity exercise tolerance in humans. Eur J Appl Physiol. 2012 Feb 10.
2. Briese E. Normal body temperature of rats: the setpoint controversy. Neurosci Biobehav Rev. 1998 May;22(3):427-36. Review. 
3. Frier BC, Locke M. Heat stress inhibits skeletal muscle hypertrophy. Cell Stress Chaperones. 2007 Summer;12(2):132-41. 
4. McArdle A, Dillmann WH, Mestril R, Faulkner JA, Jackson MJ. Overexpression of HSP70 in mouse skeletal muscle protects against muscle damage and age-related muscle dysfunction. FASEB J. 2004a Feb;18(2):355-7.
5. McArdle F, Spiers S, Aldemir H, Vasilaki A, Beaver A, Iwanejko L, McArdle A, Jackson MJ. Preconditioning of skeletal muscle against contraction-induced damage: the role of adaptations to oxidants in mice. J Physiol. 2004b Nov 15;561(Pt 1):233-44. Epub 2004 Aug 26.
6. Pumpa KL, Fallon KE, Bensoussan A, Papalia S. The effects of Lyprinol(®) on delayed onset muscle soreness and muscle damage in well trained athletes: a double-blind randomised controlled trial. Complement Ther Med. 2011 Dec;19(6):311-8.
7. Touchberry CD, Gupte AA, Bomhoff GL, Graham ZA, Geiger PC, Gallagher PM. Acute heat stress prior to downhill running may enhance skeletal muscle remodeling. Cell Stress Chaperones. 2012 May 17. [Epub ahead of print]