The most significant finding of the present study was that the increase in VO2
max was significantly greater in the CM group than the CHO or PLA groups. The average increase in absolute VO2
max for the CM group was 12.5% higher than baseline levels, a twofold improvement over the increase found in the CHO and PLA groups. The average absolute VO2
max (L/min) increase for all subjects and treatment groups combined was 9.2% over the 4.5
wk training period, which is in agreement with other investigations of aerobic training and VO2
max improvements using a similar time period [28
It has been established that the primary determinants of VO2
max are an increased ability of the cardiovascular system to transport oxygen to the working skeletal muscle, and the improved ability of the muscle to utilize the delivered oxygen. The former is a result of increased stroke volume, which improves cardiac output; the latter is determined by the increases in oxidative enzymes and mitochondrial content [1
]. We measured the activity of two key oxidative enzymes that are indicative of muscle oxidative capacity, CS and SDH. Both are found in the mitochondria and are key enzymes of the Krebs cycle, and each has been demonstrated to increase in response to endurance training [3
].We also measured total protein content of the transcription coactivator peroxisome proliferator-activated receptor γ
) as a marker for increased mitochondrial biogenesis. PGC-1α
is a transcriptional coactivator of transcription factor PPARγ
, and together, they regulate the expression of genes that encode mitochondrial proteins. An acute bout of exercise or stimulated skeletal muscle contraction induces an increase in both PGC-1α
mRNA and protein in skeletal muscle [31
], and it has been shown that increased PGC-1α
activation and total protein amount leads to increased mitochondrial biogenesis [24
In the present study, we demonstrated that the activity of CS and SDH, and the total protein content of PGC-1α
increased significantly in response to 4.5
wks of training. However, no significant treatment differences in these measures were detected. There was a slight but nonsignificant trend for a greater increase in CS and SDH activity in CM compared to CHO and PLA. It may be that the training period was not long enough to detect any potential differences that could emerge in response to chronic nutritional supplementation. Thus, our results suggest that the greater VO2
max improvements with CM supplementation are most likely due to cardiovascular adaptations rather than increases in oxidative enzymes or in mitochondrial biogenesis.
As mentioned previously, endurance training leads to an adaptive increase in cardiac output, and this increase is due to augmented stroke volume [1
]. While we did not measure these variables in the present study, our results suggest that the significant improvement in VO2
max in the CM group is likely due to increased stroke volume and cardiac output, which is likely due to increased plasma volume. Plasma volume expansion is a hallmark of aerobic endurance training [35
] and is directly associated with increased plasma albumin content. Increased albumin in the plasma causes water to be retained in the vasculature due to increases in the colloid osmotic pressure gradient [36
]. Hepatic albumin synthesis has been shown to increase in response to endurance exercise training [38
]. Moreover, plasma albumin content was reported increased 23
h after an acute bout of cycling exercise when CHO+PRO supplementation was provided postexercise compared to placebo [40
]. These results, along with the findings of the present study, suggest that hepatic albumin synthesis may have been increased to a greater extent in the CM group compared to the CHO or PLA groups and contributed to the significantly greater increase in VO2
max in the CM group.
Okazaki and colleagues [16
] recently demonstrated that CHO+PRO supplementation provided immediately after daily cycling exercise training in older male subjects increased stroke volume and plasma volume compared to a placebo group. Their subjects cycled for 60
d/wk for 8
wk at 60–75% VO2
peak and ingested either CHO+PRO or placebo immediately postexercise each session. VO2
peak increased 3.3% in the control group and 6.8% in the CHO+PRO group, with significant stroke volume and plasma volume increases only found in the CHO+PRO group [16
]. In the present study, we extend the findings of Okazaki and colleagues [16
] by demonstrating that the effect of nutritional supplementation on VO2
max increases is nutrient specific. In comparing CM against an isocaloric CHO only supplement and a placebo, we have shown that the increased VO2
max response is not due to simply providing calories postexercise. In the present study, the VO2
max increase in the CHO and PLA groups was not significantly different. Thus, these results suggest that the benefit from a CHO+PRO or CM supplement in improving VO2
max is due to the combined ingestion of carbohydrate and protein. However, we cannot rule out the possibility that a supplement composed of protein alone would not have the same effect.
In addition to well-documented increases in VO2
max with training, it is known that lactate threshold improves with endurance exercise training of moderate to high intensity [41
]. In the current study, LT improved significantly over the 4.5
wks of training although there were no significant treatment differences detected (). It has been shown that the respiratory capacity of the muscle is the key determinant of LT [42
]. Given that muscle oxidative enzyme activity and PGC-1α
content increased significantly over time without demonstrating a treatment effect, it would be expected that LT would follow a parallel pattern. Therefore, the results suggest that while LT is increased by exercise training in parallel with muscle oxidative capacity, it likewise may not be affected by nutritional supplementation.
The other key finding of the present study was that body composition improvements, represented by a calculated lean and fat mass differential, were significantly greater in the CM group than the CHO group. Compared to the CHO group, the CM group lost more fat mass and gained more lean mass measured in the whole body, as well as in the trunk region only (P < .05). While these differentials were also greater for CM compared with PLA, the differences were not significant.
It is well established that resistance exercise training induces significant gains in lean mass, whereas endurance exercise training is not associated with large increases in lean mass or gains in muscular strength [43
]. A previous investigation comparing the effects of aerobic and aerobic + resistance training showed that the aerobic + resistance group increased lean mass in arm, trunk, and total body regions, and the aerobic only group increased lean mass in trunk region only [44
]. However, the aforementioned investigation did not use supplementation. The body composition improvement with CM is also in agreement with the findings of Josse and colleagues [22
], who recently demonstrated significantly greater muscle mass accretion, fat mass loss, and strength gains with milk supplementation compared to soy and CHO after a 12-wk resistance training program [22
]. Therefore, the findings of our study are in line with what is reported in the literature for exercise mode-dependent body composition changes.
As shown in , all groups demonstrated significant changes over time in whole-body lean mass, trunk fat mass, and legs lean and fat mass, and the CM group demonstrated a significant treatment effect compared to CHO when whole body and regional differentials were calculated (Figures , , and ). The whole body and trunk differentials for PLA were slightly greater than CHO although not significantly different from either CM or CHO. The lack of difference in the PLA treatment from CHO suggests that a component of the CM treatment facilitated the significant body composition change, since simply supplementing with an energy-containing supplement (CHO) did not have a significant effect compared to PLA. In fact, a slight, nonsignificant increase in fat mass in the legs region was detected with CHO, whereas fat mass of the legs decreased in CM and PLA during the training period. To our knowledge, no evidence exists in the literature to suggest that postexercise CHO supplementation would mediate this type of change, given that the subjects' diets were not standardized and controlled during the study. However, this finding further underscores the difference in supplementing with a CHO+PRO-containing supplement versus calories from CHO alone in facilitating body composition changes.
There are two possible explanations for the difference found with the CM treatment compared to CHO: first, the availability of amino acids (AAs) in the milk for anabolism and muscle mass accretion, and second, a fat-loss promoting effect of dairy calcium and protein. It is known that AAs, along with a permissive amount of insulin, are required for muscle protein accretion to occur in response to exercise [45
]. The CHO treatment would increase plasma insulin levels and provide glucose as an energy and glycogen-synthesizing substrate, but would provide no AAs for the synthesis of new muscle protein. Thus, AAs availability from the milk proteins whey and casein provided substrate for this adaptive process. In addition, Zemel [47
] have shown that the increased consumption of dietary calcium is associated with reduced adiposity and greater weight loss in energy restricted diets. Moreover, the fat and weight loss effects were greater when the source of the dietary calcium was from dairy products rather than a calcium supplement [47
]. Additional evidence that the dairy component of the CM treatment likely underlies some of the body composition changes is found in the resistance training study of Hartman and colleagues [21
], who demonstrated that fat mass decreased, and lean mass increased, in groups provided either milk, soy, or CHO postexercise but that milk significantly promoted increased hypertrophy compared to soy and CHO [21
]. Another well-known benefit of dairy calcium consumption is improved bone mineral density. We did not detect treatment or time differences in BMD (); however, this is not surprising, given the relatively short duration of the training program and the lack of a resistance training component. Taken together, these data suggest that the dairy component of the CM treatment was instrumental in facilitating the fat mass changes compared to the CHO and PLA groups, while the AAs from milk proteins provided substrate for lean mass accretion in the present study.
There are several limitations to the present study. First, the subjects' normal diets were not controlled nor standardized for the majority of the training period. Although the diets were recorded and replicated for 3 days each week as described above, there could have been within and between-subject variations in the amount of protein, calcium, and total caloric intake on the nonrecorded days during the training period which could have influenced the adaptive response. Second, CM contains many other micronutrients and flavonoids in addition to the major macronutrients and calcium. However, the possible effects of these additional components on the training adaptations reported here are not known at this time. Third, the taste and appearance of the three treatments were different. However, the subjects were not aware of what the three treatments were, and since they only ingested the treatment for which they were randomized for the entire study period, they did not taste any of the other treatments. Fourth, we did not match the supplement dosing to each individual's body weight, but stratified the amount of supplement for each dose according to body weight ranges. We have previously shown that supplementation with ~1.0
g of CHO and ~0.3
g of PRO per kg body wt postexercise will substantially increase muscle glycogen synthesis and recovery from exercise [14
]. In the present study, providing supplement based on a weight range represented a more realistic and practical approach. Finally, while we propose that the greater increase in VO2
max in the CM group is likely due to albumin synthesis, we did not measure plasma volume or plasma albumin and, therefore, cannot say with certainly that this is the reason for the VO2
max differences. Further investigation is necessary to expand upon these results and elucidate the mechanisms of the greater adaptive response.