To the best of our knowledge, this was the first study to investigate the effects of chronic betaine supplementation in conjunction with supervised training on body composition and performance in females. The resistance training protocol promoted improvements in all body composition, muscle growth, and performance variables as evidenced by the significant main effects of time. The major findings of this study were that betaine supplementation enhanced body composition outcomes compared to resistance training alone, but not strength performance or rectus femoris muscle thickness.
Betaine supplementation has been shown in pigs to enhance muscular fatty acid uptake and oxidation  and to inhibit lipogenesis . Despite substantial evidence demonstrating betaine enhances muscle growth and reduces fat mass in animals , research examining body composition outcomes in humans is limited . In the present study betaine supplementation improved body composition by enhancing reductions in fat mass. These results are in agreement with our previous study whereby 6 weeks of betaine supplementation reduced fat mass and increased lean mass in resistance trained men . In contrast, Schwab et al.  reported no improvements in body composition with 12 weeks of betaine supplementation in sedentary obese men and women. Discrepancies in these results may be attributed to the inclusion of exercise in our studies, whereas subjects in Schwab et al. were sedentary and instructed not to change their activity.
We also hypothesized that betaine would enhance resistance training induced increases in lean mass. While there were no statistically significant differences in increases in lean mass between groups, effect sizes slightly favored the betaine group. Differences in lean mass outcomes between the present study and our previous study  may be due to dietary factors. The International Society of Sports Nutrition recommends individuals engaged in resistance training consume 1.4 to 2.2 g/kg protein per day  and recent studies suggest that protein intakes of at least 1.8 and up to 3.1 g/kg per day may be required to offset reductions in lean mass during periods of restricted energy intake [40, 41]. Subjects in the present study consumed approximately 27 ± 6.8 kcal/kg/day, which is well below World Health Organization  recommendations of 35 kcal/kg/day for physically active young women. Given the caloric deficit and lower protein intake 1.3 ± 0.35 g/kg/day in the present study, it is possible that differences in lean mass may have reached significance with a higher protein intake. In partial support of this hypothesis, Lawrence et al.  reported a significant positive interaction between betaine supplementation and protein intake on lean mass outcomes in pigs; however, future studies are necessary to verify this hypothesis.
A second hypothesis that may explain discrepancies in lean mass outcomes between this study and our previous study in males  is a result of gender differences in methyl-metabolism and tissue betaine contents. Plasma betaine concentrations are under homeostatic control and are influenced by dietary betaine intake as well as by betaine-homocysteine methyltransferase (BHMT), which utilizes a methyl group from betaine to catalyze the transmethylation of homocysteine to methionine . BHMT activity is suggested to play a key role in determining whether betaine is stored as tissue osmolyte or metabolized to provide methyl groups. Administration of estradiol and corticosteroids have both been shown to increase BHMT activity , which may explain why females typically present with lower plasma betaine and homocysteine than males . To our knowledge human data regarding gender differences in betaine tissue contents are not yet available. However, Slow et al.  reported significant differences in tissue betaine content between genders, with female mice skeletal muscle containing approximately 42% less betaine than male mice. If more betaine is metabolized in the transmethylation of homocysteine, less plasma betaine will be available for tissue uptake. As a result, lower skeletal muscle betaine concentrations in females, despite an increased stimuli for uptake as a result of the imposed metabolic stress of exercise, may have influenced the osmotic/hypertrophic effects of betaine supplementation in the current study. Lending support to this hypothesis, the results from a pig study showed that male pigs fed diets supplemented with 1 g/kg betaine/feed more efficiently converted feed into body weight gain and had greater average reduced fat depths than females ; however, further research is necessary to establish differences in betaine tissue content and skeletal muscle uptake in humans.
Betaine has been shown in vitro to promote myotube differentiation and hypertrophy by increasing IGF-1 mRNA and IGF-1 proteins as well as activating the mitogen activated protein kinase (MAPK) pathways [47, 48]. In humans 2 weeks of betaine supplementation was reported to enhance Akt signaling and downstream p70 S6K phosphorylation . Based on this evidence we hypothesized betaine would enhance rectus femoris muscle growth during the resistance training program. Our results do not support this hypothesis, as there were no differences between groups for increases in muscle thickness. These results are in partial agreement with our previous study where we found 6 weeks of betaine supplementation increased arm, but not leg lean cross sectional area . The training program in the present study was implemented as it was previously shown to prioritize lower body hypertrophy . Unfortunately, the maximal depth of the Chison Ultrasound probe is only 7.4 cm and in preliminary pilot studies this depth was not sufficient enough to measure the entire lateral quadriceps in about 33% of pilot subjects. Rather than turn potential subjects away, we decided a priori to measure only the rectus femoris muscle. It is possible that had thigh CSA or lateral quadriceps muscle thickness been measured the outcome would have been different; however, this is speculative and future research is necessary.
The effects of betaine supplementation on metrics of strength and power performance are ambiguous: a recent systemic review reported that of 7 studies published to date assessing strength and power performance, only two studies have reported positive improvements . In the present study increases in vertical jump and bench press and back squat 1 RM occurred without any significant differences between groups. These results are similar to our previous study whereby betaine supplementation did not result in greater improvements in bench press or back squat 1 RM between groups . In contrast, other studies that have employed isokinetic dynamometry have reported improvements in measures of power and force production with betaine supplementation [9, 11]. While 1 RM testing a valid and reliable test of force production that is specific to resistance training and sporting performance, it is possible that a familiarization effect took place over the course of the present study that may have confounded the results. Although we provided subjects with three familiarization sessions, Seo et al.  reported slight improvements in lower body 1 RM in female subjects over the course of 4 testing sessions.
The principal of specific adaptations of imposed demands may have also influenced the performance results. In the present study the training program was designed to optimize hypertrophy, especially in the lower body. As a result, heavy loads (> 85% 1 RM) and ballistic movements were not incorporated. Given that strength and power adaptations are maximized with heavier loads compared to higher volumes , future betaine training studies that include higher intensities and train muscular contractile velocity should be conducted to fully elucidate the effects of betaine supplementation on force and power output.
Since most studies that have reported improvements in performance have employed exercise tests that involved repeated higher intensity efforts [6, 20], therefore we previously hypothesized that betaine may be most ergogenic in testing and training protocols that impose a high metabolic demand . Metabolic stress increases the cellular uptake of betaine which results in increased cytoplasmic osmolality, biopolymer hydration, and helps to maintain biochemical function during stress by protecting ATPase and myosin heavy-chain proteins again urea denaturation , attenuating reductions in the affinity of Ca2+ for troponin , and defending citrate synthase against thermodenaturation . While strength-endurance was not specifically tested in the present study, a trend for greater total weekly volumes was found, with effect sizes favoring the betaine group in 4 of the 7 weeks as well as overall. These results are in agreement with our previous study, where we found betaine supplementation increased bench press work capacity in trained men only during the higher volume meso-cycles , and lend evidence to the hypothesis that betaine may be most ergogenic during higher volume resistance training protocols.
We hypothesized that betaine may exert ergogenic and hypertrophic effects by increasing intracellular hydration and thereby providing a more hospitable environment for excitation contraction coupling and protein synthesis. The results of the present study do not support this hypothesis, as there were no differences between groups found for total, intracellular, or extracellular water content; however, caution should be taken when interpreting the compartmental water results. First, we were unable to distinguish between compartments of intracellular water, and therefore cannot make any conclusions directly as to the effects of betaine on intramuscular hydration in particular. Second, although there were no differences between baseline and post-training nutritional status or urine specific gravity, and subjects were instructed to mimic their pre- and post-testing diets and not consume food or liquids for 3 h prior to testing, it is possible that small fluctuations in diet (sodium and fiber intake, in example) may have confounded the results. Future studies with more sophisticated equipment and strict dietary controls are required to properly assess the effects of betaine supplementation on compartmental water.