The results of the study suggest that in young, recreationally active men, calcium caseinate supplementation may lead to larger increases in maximal oxygen uptake and to greater improvements in body composition than carbohydrate supplementation, when combined with endurance training. However, the study found no evidence that these were accompanied by greater improvements in muscle oxidative capacity, hematological factors, or endurance performance with protein relative to carbohydrate supplementation.
Overall, the observed changes in VO2max were directionally as expected, as it is well-established that endurance training increases maximal oxygen uptake. The greater increases in VO2max in the PRO group relative to the CON group were also in line with the original hypothesis of the researchers. These greater increases were accompanied by significant gains in lean body mass in the PRO group (which were unsurprising, considering the significant increase in protein intake in this group), which could reflect enhanced remodelling of muscle proteins, including mitochondrial-related proteins/enzymes or endothelial and smooth muscle cells within capillaries. These may, in turn, be related to the larger improvements observed in VO2max in the PRO group.
To test whether the greater gains in VO2max in the PRO group were a result of increased mitochondrial density or function, the researchers measured the activity levels of two oxidative enzymes (citrate synthase and cytochrome C oxidase). While no differences between the groups were found, muscle oxidative capacity can’t necessarily be excluded as a potential mechanism for enhancing improvements in VO2max, as the study may have been underpowered or too short in duration to detect differences in these outcomes. This point is also supported by the fact that the differences between groups in citrate synthase and cytochrome C oxidase activity seem to be increasing over time, which suggests that a larger sample size and/or longer study duration could lead to the differences reaching statistical significance.
Interestingly, the greater improvements in VO2max in the PRO group did not result in greater improvements in endurance performance, as compared to the CON group. This is surprising in that VO2max is considered to be the single best criterion of aerobic fitness, so it would be expected that a significantly larger increase in VO2max in the PRO group would also result in a significantly greater improvement in endurance performance. Similar to the lack of changes in oxidative enzyme activity, the lack of significant differences between the two groups in endurance performance may have been due to the study being underpowered and/or too short in duration to detect such differences.
Since blood concentrations of erythrocytes and hemoglobin are significant limiting factors in oxygen transport during exercise, and contribute to the exercise-induced increase in peak oxygen uptake, it was assumed that improvements in VO2max would be accompanied by hematological adaptations in terms of larger changes in erythrocytes, hemoglobin, and hematocrit in the PRO group. However, the changes in these outcomes were not different between the PRO and CON groups, which suggests that the differences observed in VO2max between the groups cannot be explained by changes in the oxygen-carrying capacity of the blood.
Some of the strengths of the study under review include that (i) all endurance training sessions were conducted under the supervision of a researcher, so compliance with the training program and accurate measurement of training volume were assured, (ii) there was a high degree of compliance with the intake of supplemental drinks, and (iii) attempts were made to assess the participants’ dietary intakes and physical activity levels.
With the above said, it’s worth pointing out a few of the study’s limitations. First, and most importantly, the prespecified primary outcomes when the study was first pre-registered (March 2018) were citrate synthase activity, protein content of specific oxidative related proteins, and fiber specific changes, which were then changed to maximal whole-body oxidative capacity and maximal oxygen consumption in March 2019, and then changed again in the published paper to only maximal oxygen consumption. This is concerning because it leaves the possibility that several outcomes were tested in an effort to “fish” for significant results, then the primary outcomes were changed a posteriori. Second, although multiple outcomes were included and several tests were performed, there were no corrections for multiple comparisons, which means that some of the significant changes detected could be false positives. Third, it seems that the sample size requirement was not estimated using a proper power calculation method, but, instead, simply based on the sample size of another trial. This reduces confidence in the results relating to the primary outcome (VO2max), as underpowered studies are more likely to return false positives in addition to false negatives.
Fourth, body composition was assessed by DXA three-compartment model, which measures lean mass, fat mass, and bone mineral density. This means that lean mass is not identical to muscle mass, but instead includes muscle mass, organs, skin, connective tissue, and body water content. As such, it’s unknown how much of the increase in lean mass can be attributed to an increase in muscle mass. Fifth, participants comprised a relatively homogenous population of young, healthy, recreationally active males, which means that it may not be appropriate to generalize these findings to other populations, such as elderly people or well-trained athletes. Sixth, the supplement consumed by the PRO group participants was calcium caseinate, which, unlike micellar casein, is digested and absorbed at a rate similar to whey protein (i.e. fast). As such, the results may not apply to people consuming much slower proteins, such as micellar casein. Finally, the exercise program involved just cycling, which means that it may not be appropriate to generalize the results to different types of endurance training, or to programs involving a combination of endurance and strength training.
The study under review suggests that, in young, recreationally active men, supplementing with protein during ten weeks of endurance training may result in greater improvements in maximal oxygen uptake and body composition than supplementing with carbohydrates. However, these improvements may not be accompanied by greater increases in endurance performance, and don’t seem to be explained by greater improvements in muscle oxidative capacity or hematological factors. In addition, the study’s limitations relating to the changes in primary outcomes, failure to correct for multiple comparisons, and generalizability issues should be taken into account when forming conclusions.