Introduction
Betaine is a fully N-methylated derivative of the amino acid glycine. Humans consume betaine from a wide variety of foods, particularly whole wheat, spinach, and seafood (9 26 ergogenic aid (2,10,18,23
There are at least 3 mechanisms through which betaine could potentially improve exercise performance. First, betaine has been reported to increase the biosynthesis of creatine by acting as a methyl donor to methionine (11 22
Second, betaine supplementation has been reported to substantially elevate levels of blood nitric oxide , as measured using the surrogate marker of nitrate/nitrite (NOx ). In fact, 7 days of betaine supplementation at 6 g·d−1 resulted in a near-threefold increase in blood NOx (28.8 ± 3.4 vs. 82.3 ± 13.2 μM) (19 Nitric oxide is a potent vasodilator that could potentially increase muscle blood flow during an acute exercise bout. An increase in muscle blood flow could theoretically improve exercise performance by augmenting both the delivery of nutrients (e.g., oxygen and glucose) and the extraction of waste products (e.g., lactate and H+ ions).
Third, betaine can act as an osmoprotectant by regulating cellular hydration and maintaining fluid homeostasis. During periods of osmotic stress, an imbalance exists between the osmotic strength of the cytoplasm of a cell and its environment, causing water to depart from the cytoplasm. Betaine restores osmotic balance, allowing the cytoplasm to retain water. Given that dehydration and hot environments are 2 (among other) causes of osmotic stress, betaine supplementation could potentially improve performance when exercising in these conditions (2
Over the past few years, investigators have examined the ability of betaine supplementation to improve moderate- to high-intensity exercise performance. Armstrong et al. (2 2 max) after prolonged running (75 minutes at 65% O2 max) in a hot environment (31.1°C) and in a dehydrated state (fluid loss equal to 2.7% of bodyweight before consuming the betaine-fluid mixture). Betaine consumption resulted in statistically nonsignificant trends toward increased mean sprint duration (21 and 16%) when compared with consuming only water or a carbohydrate-electrolyte beverage, respectively. Hoffman et al. (18 p < 0.05) in the squat exercise (75% 1 repetition maximum [1RM]) on day 7 of betaine supplementation compared with subjects supplementing with a placebo. A similar trend for increased squat repetitions between the 2 groups (p = 0.06) was noted on day 14. However, no differences between groups existed regarding the number of repetitions performed in the bench press exercise (75% 1RM), vertical jump power, bench press throw (30% 1RM) power, or performance on the Wingate anaerobic power test. Lee et al. (23 p < 0.05) vertical jump power, isometric squat force, bench press throw (30% 1RM) power, and isometric bench press force. However, betaine supplementation failed to improve bench press (85% 1RM) repetitions, squat (85% 1RM) repetitions, or squat jump (30% 1RM) power.
To summarize, the results to date regarding betaine supplementation are generally positive but somewhat mixed. Clearly, more work is needed to elucidate the ergogenic effects of this nutrient. Thus, the purpose of this investigation was to examine the effects of a 14-day supplementation of betaine at a daily dosage of 2.5 g on exercise performance, muscle tissue oxygen saturation (StO2 ), and associated biomarkers in resistance trained men. It was hypothesized that betaine supplementation would improve measures of exercise performance, possibly by enhancing blood flow to the exercised muscles (measured indirectly using near infrared spectroscopy [NIRS]).
Methods
Experimental Approach to the Problem
Using a sample of resistance trained men, we used a double-blind, crossover design involving 14 days of betaine or placebo intake, with a 21-day washout period, to test the effects of betaine on exercise performance, blood lactate, blood malondialdehyde (MDA), blood NOx , and StO2 using NIRS. Our rationale for the measurement of blood NOx and StO2 was based on the hypothesis that betaine may increase blood flow via a nitric oxide –related mechanism, which theoretically could result in an ergogenic benefit (3 32 x is merely a surrogate marker of nitric oxide , it is commonly measured in academic research (5 6 7,29,30 14 blood flow in the same manner as does flow-mediated dilation via ultrasound technology. These considerations may be regarded as limitations of the present design.
Subjects
Thirteen resistance trained men participated in this study. The subjects were not elite level strength trained athletes but did regularly (e.g., 3–5 d·wk−1 ) perform both free weight and machine resistance exercise and could be classified as “recreationally active” resistance trained men. The subjects were nonsmokers and did not have any cardiovascular or metabolic problems that might have affected their response to treatment. The subjects did not use any performance-enhancing dietary supplements during the testing period (including creatine). The subjects who reported using performance-enhancing dietary supplements were required to stop using these for a period of 2 weeks before beginning baseline testing. Health history, drug and dietary supplement usage, and physical activity questionnaires were completed by all the subjects to determine eligibility. Heart rate (HR) and blood pressure were recorded after a 10-minute period of quiet rest. Height, weight, circumference measures, and body fat percentage (determined via a 7-site skinfold measurement) were recorded and used as descriptive characteristics. A maximal test in the bench press exercise was performed on a machine device (Hammer Strength™). The subjects were given multiple attempts as they worked up to their maximal effort, with 2–4 minutes of rest given between attempts. The highest load lifted in good form was recorded as their 1RM. The guidelines from the National Strength and Conditioning Association were followed, and all testing was overseen by a Certified Strength and Conditioning Specialist.
Before any testing, each subject was informed of all procedures, potential risks, and benefits associated with the study. All the procedures were performed in accordance with the guidelines of the American College of Sports Medicine with regards to human experimentation and approved by the University Human Subjects Review Board (H10-44). The subjects provided both verbal and written consent before participating. Subject characteristics are presented in Table 1 .
Table 1: Descriptive characteristics of 13 resistance trained men.*†
Procedures
Within 1 week of the initial screening visit, all subjects performed the entire exercise test protocol as described below, consuming water only (as opposed to the conditions described below). This served as a familiarization session to fully acclimate the subjects to the exercise tests.
Independent Variable (Conditions)
The subjects were randomly assigned in a double-blind manner using a crossover design to consume betaine (2.5 g of betaine powder [BetaPower™: betaine anhydrous; Danisco, Tarrytown, NY, USA] mixed into 500 mL of Gatorade®) or a placebo (500 mL of Gatorade®). This is the same amount of betaine used in 2 recent investigations involving resistance exercise performance (18,23 dietary supplements at a dosage of 2–2.5 g. Therefore, we believe that our dosage is both scientifically appropriate and practically relevant. The subjects were instructed to ingest 250 mL twice daily for the 14-day period. A 21-day washout period was included between betaine and placebo conditions. Testing (as described below) was conducted before and after each 14-day treatment period. Therefore, the subjects performed the entire testing protocol a total of 5 times (familiarization, prebetaine and postbetaine, preplacebo and postplacebo).
Testing (Second-Fifth Laboratory Visits)
For each test day, the subjects reported to the laboratory in a 10-hour fasted state to perform the exercise performance tests. The time of testing was similar for each subject on all testing days. After the collection of a fasting blood sample, subjects' upper torso circumference was measured (at the level of the nipple), and subjects rated their subjective assessment of muscle pump using a visual analog scale. The subjects then consumed either 2.5 g of betaine powder mixed in 500 mL of Gatorade® (on day 14 of the betaine supplementation period) or 500 mL of Gatorade® only (on all other visits–including the prebetaine visit). Thirty minutes after intake of their assigned condition, the subjects began the exercise test. The subjects were instructed to warm up (including light stretching) for a 10-minute period before performing the exercise tests. The subjects did not consume any food during the testing period, but water was provided ad libitum during session 1. The amount of water consumed was recorded, and the subjects were asked to match this intake during all subsequent test sessions. Despite this attempt to control fluid intake, hydration status of subjects was not measured. This may be considered a limitation of this work.
The performance protocol included the following 5 tests, completed in the order indicated below, with 3–5 minutes separating each test:
(a) Lower-body muscular power using a countermovement, bodyweight-only vertical jump: This test was conducted using a Vertec® device. The best attempt of the 3 jumps was recorded and used in the data analysis.
(b) Upper-body muscular power using a bench press throw (ProSpot® device): After a warm-up featuring 3 practice throws using 10% of their predetermined 1RM, the subjects performed 3 throws using 30% of their 1RM. Ninety seconds of rest was provided between each throw. The best attempt of the 3 throws was recorded and used in the data analysis. A detailed description of this assessment is provided elsewhere (13
(c) Lower-body maximal isometric force using a modified Hammer Strength™ leg press: The seat of the leg press device was adjusted for each subject so as to create a knee angle of 120°. The subjects folded their arms across their chest and maintained this position throughout the exercise test. The subjects warmed up by pressing against the footplate twice at 50 and 75% of perceived maximum effort. After this warm-up, the subjects pressed against the footplate as hard and as quickly as possible for a 3- to 5-second duration, interspaced with 60 seconds of rest. The best attempt of the 3 presses was recorded and used in the data analysis. A detailed description of this assessment is provided elsewhere (28
(d) Upper-body maximal isometric force: Data were obtained in a bench press position using a customized force plate and power rack design. The power rack had 1-in. hole spacing for individual bar height adjustments. The subjects positioned themselves on the bench at a location that aligned their midsternum with the bar. The subjects were instructed to grip the bar at a location that positioned the upper arm parallel to the floor and created a 90° angle about the elbow joint. From this position, the corresponding grip width and fixed bar height were recorded and reproduced for all testing sessions. The subjects warmed up by pressing against the bar twice at 50 and 75% of perceived effort. After this warm-up, the subjects pressed against the bar as hard and as quickly as possible for a duration of 3–5 seconds, interspaced with 60 seconds of rest. Data were sampled at 1,000 Hz and channeled though a 12-bit analog-to-digital converter (DAS1200Jr; Measurement Computing). Force data were smoothed using a digital low-pass filter (cutoff frequency 30 Hz) and analyzed for peak force using Datapac 5.
(e) Upper-body muscular endurance: After the upper-body maximal isometric force test, a sensor was placed on subjects' dominant-arm anterior deltoid muscle for a measure of StO2 using NIRS, as described below. The subjects then performed 10 sets in the Hammer Strength™ supine bench press exercise using a load equal to 50% of their 1RM. This same intensity of resistance exercise has been used in many previous studies and also in 2 studies incorporating NIRS (4,30
Within 1 minute of the conclusion of the final set of the bench press exercise test, a second blood sample was taken. After the blood sampling, subjects' upper torso circumference was measured, and subjects rated their subjective assessment of muscle pump using a visual analog scale. This marked the conclusion of the laboratory visit.
Near Infrared Spectroscopy
Muscle tissue oxygen saturation was measured continuously (during both each set and each rest period) during the bench press protocol using an InSpectra™ Tissue Oxygenation Monitor (Hutchinson Technology, Hutchinson, MN, USA). This system uses NIRS (calibrated wavelengths of near infrared light) to noninvasively illuminate the tissue below a sensor that is placed on the skin surface. This device quantifies the ratio of oxygenated hemoglobin to total hemoglobin in the microcirculation of the volume of illuminated tissue. We have determined in pilot work that the system is most sensitive when the sensor is applied to the anterior deltoid muscle (as opposed to the pectoralis major or pectoralis minor muscle).
Our rationale for using this instrument in the present design was based on the potential ability of betaine to stimulate an increase in blood flow (via any mechanism). If effective at doing so, then the amount of oxygen saturation at the start of each set of exercise may be greater, and the amount of oxygen saturation at the end of each set of exercise may be less (indicating greater tissue oxygen consumption). Based on this rationale, we recorded the exact starting oxygen saturation (StO2 start) and ending oxygen saturation (StO2 end) for each of the 10 sets of exercise. The difference in oxygen saturation between the onset and the conclusion of each set (StO2 difference) was also calculated.
Blood Sampling and Biochemistry
Venous blood samples (5 ml) were taken from the subjects (in a fasted and rested state) via a needle and Vacutainer (Franklin Lakes, NJ, USA) before starting exercise and within 1 minute of the conclusion of the muscular endurance bench press test (before and after each 14-day period of betaine and placebo supplementation). Single samples were immediately analyzed for whole-blood lactate using an Accutrend analyzer (Roche Diagnostics, Mannheim, Germany). The remainder of the whole blood was processed for plasma and stored at −70° C until analysis. The following assays for nitrate/nitrite and MDA were performed in duplicate.
Nitrate/nitrite was analyzed in plasma using a commercially available colorimetric assay kit (Caymen Chemical, Ann Arbor, MI, USA) according to the procedures provided by the manufacturer. After being thawed, plasma samples were centrifuged at 10,000 g for 3 minutes in a refrigerated centrifuge (4°C). After the addition of a nitrate reductase cofactor to each diluted sample, nitrate reductase was added, and the mixture was incubated for 3 hours to allow for the full conversion of nitrate to nitrite. Greiss reagent was then added, which converts nitrite into a deep purple azo compound. The absorbance was detected photometrically at 540 nm. Quantification was performed with a calibration curve. The coefficient of variation for this assay in our laboratory is <8%. The detection limit, as per the manufacturer, is ≥2.5 μM.
Malondialdehyde was analyzed in plasma following the procedures of Jentzsch et al. (20
Dietary Records and Activity
All the subjects were instructed to consume a low nitrate diet (no cured meats, green leafy vegetables, beets, berries); otherwise, the subjects were instructed to maintain their normal diet throughout the study period and record dietary intake during the day before each test day. The subjects were asked to mimic this intake during the day preceding each subsequent test day. All diet records were analyzed for total calories, protein, carbohydrate, fat, vitamin C, vitamin E, and vitamin A (Food Processor SQL, version 9.9, ESHA Research, Salem, OR, USA). The subjects were asked to maintain their usual exercise routine throughout the entire study period (during both placebo and betaine phases) but to refrain from strenuous physical activity during the 48 hours that preceded each test day—in an attempt to allow for full recovery and to minimize muscle soreness that may have been present because of their usual exercise training.
Statistical Analyses
All dietary data, performance data, mean HR data, and mean rating of perceived exertion data were analyzed using a 2 (condition) × 2 (pre/post intervention) repeated measures analysis of variance (RMANOVA). Blood lactate, NOx , MDA, subjective muscle pump, and circumference data were analyzed using a 2 (condition) × 2 (pre/post intervention) × 2 (time) RMANOVA. The StO2 data (start, end, difference) were first analyzed using a 2 (condition) × 2 (pre/post intervention) × 10 (set number) RMANOVA. The data were then collapsed by set number and analyzed using an ANOVA to compare conditions from preintervention to postintervention without considering set number. Post hoc testing was performed where appropriate. In addition, paired t -tests were used to compare certain pre and postbetaine performance measures. Data were first tested for normality. The outcome data are presented as mean ± SEM . Subject descriptive characteristics are presented as mean ± SD . Statistical significance was set at p ≤ 0.05.
Results
Compliance to Treatment and Dietary Intake
All the subjects successfully completed all the components of the study. No adverse effects were noted with either betaine or placebo supplementation. All the subjects tolerated treatment with betaine well and reported no aversion to supplementation. No interaction or main effects were noted for dietary data with regard to any measured variable (p > 0.05). Dietary data are presented in Table 2 .
Table 2: Dietary data of 13 resistance trained men receiving placebo or betaine in a crossover design.*†‡
Performance Measures
Considering the RMANOVA, no statistically significant differences were noted between conditions for any measured exercise performance variable (p > 0.05). However, the subjects performed more repetitions (p = 0.01) and a greater total volume load (p = 0.02) in the bench press exercise after betaine treatment (paired t -tests), with values increasing approximately 6.5% from preintervention to postintervention. No difference was noted in the mean HR or the mean perceived exertion over the 10 sets (p > 0.05). Data are presented in Table 3 .
Table 3: Exercise performance data of 13 resistance trained men receiving placebo or betaine in a crossover design.*†‡
Biochemical Measures
With the exception of a time effect noted for blood lactate (p < 0.0001), no statistically significant effects were noted for any measured variable (p > 0.05). Although not of statistical significance (p = 0.14), subjects supplementing with betaine experienced an attenuated exercise-induced increase in blood lactate (210% increase) compared with subjects supplementing with a placebo (270% increase). The increase in blood lactate levels from pre to postexercise during placebo supplementation essentially did not change from preintervention to postintervention. Blood NOx was slightly lower postintervention as compared with preintervention (p = 0.06) for both betaine and placebo conditions. Also, MDA did not change during either supplementation period. Data for all biochemical measures are presented in Table 4 .
Table 4: Biochemical data of 13 resistance trained men receiving placebo or betaine in a crossover design.*
Muscle Pump and Circumference
Perceived muscle pump significantly increased (p < 0.0001) from pre to postexercise both preintervention and postintervention for both conditions. With the exception of this time effect, no statistically significant effects were noted for either perceived muscle pump or upper torso circumference (p > 0.05). Data are presented in Table 5 .
Table 5: Circumference and perceived muscle pump data of 13 resistance trained men receiving placebo or betaine in a crossover design.*
Muscle Tissue Oxygen Saturation
When considering the 2 (condition) × 2 (pre/post intervention) × 10 (set number) ANOVA, the following was noted: For StO2 start, only a set number (p < 0.0001; set 1 lower than all other sets) and a pre/postintervention effect (p = 0.008; pre lower than post) was noted. No other changes of statistical significance were noted. For StO2 end, only a condition effect was noted (p = 0.01; betaine lower than placebo). No other changes of statistical significance were noted. For StO2 difference, as expected based on the condition effect for StO2 end, a condition effect was noted (p = 0.01; betaine higher than placebo). No other changes of statistical significance were noted. Data are presented in Table 6 .
Table 6: Muscle tissue oxygen saturation data for 10 sets of bench press exercise in 13 resistance trained men receiving placebo or betaine in a crossover design.*†
When removing set number from the model and only considering the condition comparison preintervention and postintervention, similar effects were apparent. That is, a pre/postintervention effect was noted for StO2 start (p = 0.009), with values higher postintervention compared with preintervention (when pooling both betaine and placebo; however, only betaine demonstrated statistical significance when individual pre-post comparisons were made [p = 0.03]). The StO2 end was lower for betaine compared with placebo (p = 0.01), which translated into a larger StO2 difference for betaine (p = 0.01). No other statistically significant changes were noted. Data are presented in Table 7 .
Table 7: Muscle tissue oxygen saturation data collapsed over 10 sets of bench press exercise in 13 resistance trained men receiving placebo or betaine in a crossover design.*†
Discussion
Our findings indicate that betaine supplementation results in a moderate (∼6.5%) increase in total repetitions and volume load when supplemented at a daily dosage of 2.5 g for 14 days. We noted no other differences in measures of exercise performance or in biochemical parameters between betaine and placebo supplementation. However, exercise-induced blood lactate accumulation was slightly lower during betaine supplementation vs. placebo supplementation (although not of statistical significance), which may have some practical application for individuals engaged in exercise bouts resulting in high lactate accumulation. From these data, it appears that betaine primarily provides benefit during exercise challenges of moderate intensity and high volume (the 10 set bench press test)—possibly the type of exercise resulting in higher lactate accumulation and alteration in cellular hydration. It is possible that the slightly lower lactate accumulation, coupled with the potential effect of maintaining cellular hydration during such repeated effort sets, may have contributed to our findings for betaine.
It should be noted that the 2.5 g·d−1 dosage of betaine provided in this study is the same as provided in 2 recent investigations involving resistance exercise performance (18,23 dietary supplements at a dosage of 2–2.5 g. The above information was considered when determining the dosage to be used in this study. It is presently unknown whether or not a higher (or lower) dosage of betaine would provide similar or greater benefits with regard to resistance exercise performance. Additionally, the betaine was delivered in a commercially available sport drink, as has been done in prior investigations using betaine for purposes of improved exercise performance (18,23
We hypothesized that StO2 values at the start of exercise would be greater after treatment with betaine. When pooling all sets, this was confirmed by a slightly higher StO2 value after 2 weeks of betaine supplementation (Table 7 ). The StO2 values at the end of exercise were also lower during betaine supplementation, which translated into a greater StO2 difference. This suggests that enhanced muscle oxygen consumption may have occurred, as previously noted (30 2 decreased with each subsequent bench press repetition, suggesting that increased repetitions would require increased muscle oxygen consumption and lead to further decrements in StO2 . In addition, decreased blood lactate accumulation may suggest that anaerobic glycolysis was de-emphasized in favor of metabolic pathways that involve oxygen consumption.
The osmoprotectant properties of betaine may have contributed to enhanced muscle oxygen consumption, assuming that the lower StO2 values at the end of exercise indicate that such a phenomenon did occur. Cells accumulate betaine to help maintain optimal cellular function in the presence of stressors such as perturbing electrolytes, urea, ammonia, and others (9,24 1 27 8 34 2 difference may reflect a more robust and optimized cellular state that allows for greater oxygen desaturation and results in greater energy production.
In comparison with our findings, 2 previous studies (18,23 dietary supplements , that certain individuals are responders to treatment, whereas others are not. The sample size for each study performed to date has been relatively small, which potentially impairs the ability to detect statistical significance. Future studies are needed to investigate the ergogenic potential of betaine, preferably with the inclusion of larger subject samples. Moreover, although our subjects were regularly performing resistance exercise (average of 4 h·wk−1 for the past 4 years), they were not elite strength-trained athletes. Although we believe that our subject pool is representative of the majority of recreationally active resistance trained men who might consider the use of betaine as a dietary supplement, we cannot state with confidence that betaine would prove beneficial for all athletes, including those who are considered to be elite. As with all dietary supplements , variability in response to treatment likely exists, requiring self-experimentation on the part of the individual to determine effectiveness.
To put our finding of an approximately 6.5% increase in total bench press repetitions in perspective, several investigations examining creatine monohydrate supplementation (for 30 days or fewer) in resistance trained men (minimum of 3 months' experience) have reported increases in this variable ranging from 26 to 39% (12,21,31 17 15,33
Our findings do not support the improved performance reported by Lee et al. regarding isometric bench press force, bench press throw power, and vertical jump power (23 18 18
Betaine supplementation had no effect on blood NOx . Moreover, our recent investigation on the effects of betaine supplementation at 6 g·d−1 for 8 days noted a similar lack of effect (data under review). Together, these data indicate that any ergogenic effect of betaine supplementation is not mediated by an increase in circulating NOx . The results of our work starkly contradict the approximately 286% increase in blood NOx reported by Iqbal (19 −1 . However, the Iqbal study was conducted using older subjects who may have had a diminished ability to generate nitric oxide (16,25 x is influenced by age.
In conclusion, betaine supplementation at a dosage of 2.5 g·d−1 resulted in a moderate increase in total repetitions and volume load. No other measure of muscle performance was impacted by betaine supplementation nor was any biochemical measure statistically different between betaine and placebo. Betaine treatment resulted in a slightly reduced albeit statistically insignificant blood lactate accumulation, and an increased StO2 difference compared with placebo treatment. One or both of these effects may help to explain the moderate increase in total repetitions performed during the bench press exercise. Although research on betaine remains in its infancy, additional well-designed investigations are needed to extend our work and the work of others who have focused on the study of this nutrient.
Practical Applications
While our data indicate that betaine supplementation at 2.5 g·d−1 for 14 days has little to no effect on muscular force and power production, it may improve performance in activities that emphasize muscular endurance. Consequently, a training effect may be observed over time, but this remains to be confirmed. Coupled with findings of increased muscular force and power production noted in other investigations, it is possible that betaine may prove effective as an ergogenic aid for certain individuals. As with all dietary supplements touted as ergogenic aids, betaine will require experimentation on the part of each individual considering use of this ingredient. Such self-experimentation, along with additional laboratory-based research, should provide additional answers regarding the ergogenic benefits of betaine supplementation.
Acknowledgments
This work was supported by Danisco and The University of Memphis. This support does not necessarily imply endorsement by the University of research conclusions.
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