Beyond brawn: can protein supplementation fuel aerobic improvement?

Maximal oxygen uptake (also known as VO_2max)^[1] is the highest rate at which oxygen can be transported and utilized by the body during intense exercise. Since VO_2max is considered to be the single best criterion^[2] of aerobic (or cardiorespiratory) fitness, and as aerobic fitness is independently associated with positive health outcomes^[3] as shown in Figure 1, many athletes and recreationally-active people aim to improve their aerobic fitness by performing endurance exercise. Indeed, endurance exercise has been shown to be effective in improving^[4] cardiorespiratory fitness, likely as a result of a number of adaptations^[5] that improve the ability of the cardiorespiratory system (heart, lungs, and blood) to deliver oxygen to exercising muscles and the ability^[6] of the muscles to utilize oxygen.

Together with endurance exercise, nutrition plays an important role in improving aerobic fitness. For example, it has been relatively well-established that carbohydrate ingestion^[7] around the time of exercise, as well as a sufficient intake of dietary carbohydrates to maintain the body’s glycogen stores, are associated with improved endurance performance. Although most of the attention has been on carbohydrates for improving endurance performance, there has also been increasing interest in the role of protein for enhancing endurance training adaptations. Research^[8] suggests that dietary protein may play a critical role in supporting recovery from endurance exercise.

Despite the fact that acute protein ingestion has shown benefits for endurance exercise recovery^[9] and performance^[10], data on the role of protein supplementation in relation to the adaptive response to chronic endurance exercise is generally lacking. To date, only a handful of small studies^[11][12][13] with important methodological limitations have investigated the effects of protein supplementation on the adaptations to endurance exercise, with conflicting results. The study under review was the first preregistered double-blind randomized trial to investigate the effects of protein supplementation on changes in VO_2max after 10 weeks of endurance training.

Maximal oxygen uptake (VO_2max) is the highest rate at which oxygen can be transported and utilized by the body during intense exercise, and the single best marker of aerobic fitness. While most studies have focused on carbohydrate ingestion for improving endurance training performance, recent research suggests that dietary protein may play an important role in promoting endurance training adaptations. However, only a handful of small studies have examined the effects of protein supplementation on endurance training-induced adaptations and aerobic fitness with chronic aerobic exercise. The study under review aimed to add to the limited body of literature by examining the effects of protein supplementation on changes in VO_2max with endurance training.

This was a preregistered double-blind randomized trial^[14] involving 40 young men between 18 and 30 years old (average age of 22 years) with an average BMI of 22.4 (BMI range between 18.5 and 25; i.e., the healthy range), and an average maximal oxygen uptake capacity (VO_2max) of 50.4 mL/kg/min, which is just under^[15] the 80th percentile for men their age. As shown in Figure 2, both age and sex matters for ranking VO_2max. The participants were healthy, injury-free, non-smokers, and did not use any medication or nutritional supplements. They were also recreationally active, performing sports on a non-competitive basis for one to four hours per week, and had no history of participating in structured endurance training programs within the last two years.

Figure 2: 80th percentile for VO_2max by age

Following two weeks of baseline testing, participants were randomly assigned to supplement with a drink containing either protein (PRO; 29 grams of calcium caseinate) or an isocaloric amount of carbohydrates (CON; sucrose and maltodextrin) post-exercise (three times per week) and pre-sleep (seven times per week), while also completing a supervised progressive endurance training protocol three times per week for 10 weeks. The training protocol involved 60 minutes of continuous vigorous-intensity^[16] (i.e. requiring a large amount of effort and causing rapid breathing and a substantial increase in heart rate) cycling, and was divided into two blocks of five weeks with one week between the blocks (week six) to perform the midterm measurements, and a final week (week 12) to perform the final measurements. In addition to the post-exercise drink, participants also consumed two slices of gingerbread immediately after exercise, containing 280 calories, 63 grams of carbohydrates, 2.4 grams of protein, and 1.2 grams of fat. Before each test day, they were provided with a standardized meal to consume in the evening.

Over the course of the intervention, participants were instructed to maintain their habitual dietary intake and physical activity patterns. Dietary intake was assessed by three-day weighted dietary intake records at baseline, week five, and week 12, while habitual physical activity and supplemental exercise were assessed using a self-administered seven-day activity recall questionnaire.

The initial prespecified primary outcomes, according to the clinical trial record history^[17], were the muscle adaptive response in terms of citrate synthase activity, protein content of specific oxidative related proteins, and fiber specific changes. These were later changed to maximal whole-body oxidative capacity and maximal oxygen consumption. However, in the published paper, the only specified primary outcome was the change in maximal oxygen consumption (i.e., VO_2max). Also, while the paper suggests that a power calculation was conducted to estimate the sample size requirement, no information was provided on the exact calculation method. Instead, it seems like the researchers aimed to have a sample size equal to or larger than the sample size used by an earlier, somewhat similar, trial^[13]. Secondary outcomes included skeletal muscle oxidative capacity, endurance performance, hematological factors, and body composition via three-compartment model DXA.

In this double-blind randomized trial, 40 recreationally-active young men received either a casein protein supplement or an isocaloric carbohydrate drink post-exercise and pre-sleep during 10 weeks of supervised endurance training. The primary outcome was the change in VO_2max between groups.

As shown in Figure 3, there were significant increases in VO_2max (which was the primary outcome) from pre- to mid-intervention (PRO: +10%; CON: +4.3%), and from pre- to post-intervention (PRO: +11%; CON: +6.1%) in both groups, with the increases being significantly greater in the PRO relative to the CON group (pre- to mid-study: p=0.017; pre- to post-study: p=0.045).

Figure 3: Changes in the primary outcome over time

In terms of body composition, lean body mass significantly increased in the PRO group from pre- to mid-intervention (+2.5%), and from pre- to post-intervention (+2.5%), but remained unchanged in the CON group. The changes at both time points for lean body mass were significantly greater in the PRO relative to the CON group. Moreover, fat mass significantly decreased in the PRO group from pre- to mid-intervention (-4.7%), and from pre- to post-intervention (-9.4%), but remained unchanged in the CON group. The changes in fat mass from pre- to post-intervention, but not from pre- to mid-intervention, were significantly greater in the PRO relative to the CON group.

With regard to the other secondary outcomes, endurance performance and muscle oxidative capacity were improved in both treatments with no differences between the groups, while any changes in hematological factors during the intervention returned to baseline levels at the end of the study, with no differences between the groups.

According to the dietary intake data, protein intake significantly increased from baseline to the end of the study in the PRO group (from 1.3 to 1.8 grams per kilogram of bodyweight), but not in the CON group (from 1.3 to 1.3 grams per kilogram of bodyweight), with the increases being significantly different between the groups.

Finally, compliance to the training program and supplementation regimen was very high (between 98% and 100%) with no differences between groups.

Ten weeks of endurance training increased VO_2max in the PRO and CON groups, with the increases being significantly greater in the PRO, relative to the CON, group at mid- and post-intervention. Also, lean body mass increased more, and fat mass decreased more in the PRO group relative to the CON group.

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 VO_2max were directionally as expected, as it is well-established^[4] that endurance training increases maximal oxygen uptake. The greater increases in VO_2max 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 VO_2max in the PRO group.

To test whether the greater gains in VO_2max 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 VO_2max, 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 VO_2max in the PRO group did not result in greater improvements in endurance performance, as compared to the CON group. This is surprising in that VO_2max is considered to be the single best criterion^[18] of aerobic fitness, so it would be expected that a significantly larger increase in VO_2max 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^[19] in oxygen transport during exercise, and contribute to the exercise-induced increase^[20] in peak oxygen uptake, it was assumed that improvements in VO_2max 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 VO_2max 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^[21], but, instead, simply based on the sample size of another trial. This reduces confidence in the results relating to the primary outcome (VO_2max), as underpowered studies^[22] 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^[23] 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.

The study under review confirms the positive impact of chronic endurance training on maximal oxygen uptake, with the average observed improvements in the two groups of around 4.3 mL/kg/min at week 10 being in line with the improvements in VO_2max with endurance training relative to no training reported in a 2015 meta-analysis^[4].

The greater improvements in VO_2max observed in the study under review with protein, relative to carbohydrate, supplementation, are in agreement with the results of some previous studies, but not all.

For example, one study^[13] in 32 young, recreationally active men and women performing 60 minutes of endurance training five times per week, reported larger increases in VO_2max with chocolate milk supplementation (providing 18-28 grams of protein per day, according to bodyweight) relative to carbohydrate supplementation. However, the participants’ diets were not controlled for the majority of the treatment, and the protein drink (chocolate milk) contained other micronutrients, in addition to the major macronutrients, which could have contributed to the greater VO_2max improvements. Another study^[12] in 16 untrained middle-aged men and women performing a progressive aerobic exercise routine three times per week for six weeks reported that participants supplementing with 20 grams of milk protein after exercise experienced greater improvements in VO_2max than participants consuming an isocaloric amount of carbohydrates. However, this study was not designed to assess changes in VO_2max as the primary outcome, which means that the results should be considered exploratory.

On the other hand, a 2018 study^[11] in 17 young runners undergoing a progressive endurance training program for 10 weeks found no differences in VO_2max with supplementation of 50 grams of whey protein per day, compared to placebo. Surprisingly, the study also found increases in lean body mass in the placebo, but not in the protein group, as well as larger improvements in endurance performance in the placebo, relative to the protein group. Moreover, a 2019 study^[24] in 60 recreationally active young men found that protein supplementation after endurance exercise and before sleep during 12 weeks of endurance training (three times per week) did not result in larger VO_2max increases in the protein, relative to the placebo, group. While this study was very similar in design to the study under review, the protein supplement used was a micellar casein (as opposed to a calcium caseinate in the study under review), which has a much slower digestion and absorption rate^[25], and which may have affected the results. The discrepancies observed in the results of different trials are likely due to methodological differences between studies, such as in sample sizes, training regimens, degree of monitoring, and the types and amounts of supplements consumed.

The study under review found greater improvements in body composition with protein supplementation relative to carbohydrate supplementation, with larger increases in lean body mass and larger decreases in fat mass in the PRO group. These results are in line with the available research^[26], which suggests that aerobic exercise can increase lean body mass, provided that it is carried out with sufficient intensity, duration, and frequency, as well as with the research^[27] suggesting that higher protein intakes (1.2-2.4 grams per kilogram of bodyweight) in exercising individuals may offer benefits in terms of an increase in lean mass and decrease in fat mass.

The study under review also found that the provided supplementation protocol improved endurance performance with endurance training, but that there were no differences between the protein and carbohydrate groups, despite the greater improvements in VO_2max and body composition in the PRO group. As mentioned above, the lack of significant differences may have been due to the study being underpowered or too short in duration. On the other hand, it is remarkable that in another study^[11] in runners, it was reported that protein supplementation did not improve endurance performance, and also that the placebo group experienced larger (approaching significance) improvements. Although baseline characteristics in regard to endurance performance were not found to be different between groups, the placebo group participants were, on average, more than 10% slower in the five kilometer treadmill trial at baseline than the protein group participants, and, even at the end of study, were still more than 6% slower than the baseline values of the protein group participants. Due to lower baseline performance, they likely had more room for improvement.

Since it is thought that the ability of muscles to use oxygen during exercise is one of the main determinants of VO_2max, the muscles’ oxidative capacity was measured in the study under review. However, no differences were detected between groups. These results are in line with the results of one of the aforementioned studies^[13], which reported that the activity of two key oxidative enzymes was not different between the chocolate milk and carbohydrate groups after 4.5 weeks of endurance training. However, this study, as well as the study under review may have been underpowered and/or too short in duration for a significant difference between groups to have been detected. Finally, while hematological adaptations have been found to play a key role^[20] in improvements in VO_2max with endurance training, no differences between groups for these outcomes were found in the study under review.

There are important methodological differences within the available research, which make it difficult to draw definitive conclusions. What can be said with relative certainty is that a sufficient intensity, duration, and frequency of endurance training should improve VO_2max, while a sufficient intake of protein during chronic endurance training will likely optimize the improvements in body composition and VO2max. Whether these improvements are the result of increases in maximal oxidative capacity, and if they may result in improvements in endurance performance, is currently unknown.

Q. How exactly is protein thought to improve endurance training performance?

Our current understanding of how protein ingestion during recovery from endurance exercise may improve subsequent performance is still limited, while the role that dietary protein has in supporting skeletal muscle remodeling has only recently started to receive scientific attention.

A number of potential mechanisms exist through which protein may improve endurance training recovery and performance. First^[28], amino acids may be oxidized and used for the generation of glucose in the liver or they can be broken down and used as a direct source of fuel by the mitochondria in muscles, although this is unlikely to meaningfully improve performance, as amino acids are not an efficient fuel source. Second^[8], protein can promote the remodeling of muscle fibers, and maintain muscle protein quality and function by removing old or damaged proteins. Third^[8], protein may stimulate myofibrillar protein synthesis and lead to a net increase in myofibrillar proteins, enabling greater muscle power output. Finally^[29], coingestion of protein with carbohydrates may promote glycogen resynthesis, helping to maintain subsequent exercise performance.

VO_2max is the highest rate at which oxygen can be transported and utilized by the body during intense exercise, and is the best criterion of aerobic fitness. While most research has focused on carbohydrate ingestion for improving endurance training performance, there is evidence that dietary protein may also play an important role in the adaptive response to endurance training. However, only a handful of small studies have investigated the impact of protein supplementation during chronic aerobic exercise on aerobic fitness. The study under review aimed to add to the limited body of literature by assessing the effects of protein supplementation on changes in VO_2max with endurance training.

According to the study under review, and of the available relevant research, endurance training can improve VO_2max, while a sufficient intake of protein during chronic endurance training will likely optimize the improvements in body composition and VO_2max. However, researchers are uncertain if these improvements are the result of increases in maximal oxidative capacity, and if they may lead to further improvements in endurance performance.