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RES E AR C H A R T I C L E Open Access
Effects of betaine on body composition,
performance, and homocysteine thiolactone
Jason M Cholewa
1*
, Monika Wyszczelska-Rokiel
2
, Rafal Glowacki
2
, Hieronim Jakubowski
3,4
, Tracey Matthews
5
,
Richard Wood
5
, Stuart AS Craig
6
and Vincent Paolone
5
Abstract
Background: This study investigated the effects of long term betaine supplementation on body composition,
performance, and homocysteine thiolactone (HCTL) in experienced strength trained men.
Methods: Twenty-three subjects were matched for training experience (4.8 ± 2.3 years) and body fat percentage
(BF%: 16.9 ± 8.0%), randomly assigned to either a placebo (PL; n = 12) or betaine group (BET; n = 11; 2.5 g/day), and
completed a 6 week periodized training program consisting of 3 two-week micro-cycles. Bench press and back
squat training volumes were recorded and changes in training volume were assessed at each micro-cycle. Fasting
urine was collected at baseline (BL), weeks 2, 4 and 6, and assayed for HCTL. Subjects were tested prior to and
following 6 weeks of treatment. Arm and thigh cross sectional area (CSA) was estimated via girth and skin fold
measurements. Body density was estimated via skin fold calipers and used to estimate BF%, fat mass (FM), and
lean body mass (LBM). Performance was assessed via vertical jump (VJ), bench press 1 RM (BP), and back squat
1 RM (BS).
Results: Arm CSA increased significantly (p < .05) in BET but not PL. No differences existed between group and
time for changes in thigh CSA. Back squat training volume increas ed significantly (p < .05) for both groups
throughout training. Bench press training volume was significantly (p < .05) improved for BET compared to PL at
microcycles one and three. Body composition (BF%, FM, LBM) improved significantly (p < .05) in BET but not PL.
No differences were found in performance variables (BP, BS, VJ) between groups, except there was a trend (p = .07)
for increased VJ power in BET versus PL. A significant interaction (p < .05) existed for HCTL, with increases from BL
to week 2 in PL, but not BET. Additionally, HCTL remained elevated at week 4 in PL, but not BET.
Conclusion: Six-weeks of betaine supplementation improved body composition, arm size, bench press work
capacity, attenuated the rise in urinary HCTL, and tended to improve power (p = .07) but not strength.
Keywords: Hypertrophy, Strength, Power, Supplementation, Trimethylglycine
Background
Betaine (trimethylglycine) is an organic osmolyte found
in many foods, including spinach, beets, and whole
grains [1]. Administration of supplemental betaine for
10–15 days has enhanced performance in several studies
but with varying results: Lee et al. [2] reported increased
power output and force production, whereas others [3,4]
reported improvements in muscular endurance but not
power. On the other hand, Del Favero et al. [5] reported
no improvements in power output, strength, or body
composition with 10 days of betaine treatment; however,
subjects were instructed to avoid training and sup-
plementation wa s ceased 5 days prior to performance
testing.
To the author’s knowledge, only two stud ies have exa-
mined the effects of betaine on body composition and
hypertrophy in humans. Betaine did not improve body
composition in obese, sedentary subjects on a 500 kcal/
day caloric deficit following 12 weeks of supplementation
[6]. Similarly, 10 days of betaine supplementation did not
improve body composition in sedentary young male sub-
jects [5]. Though research is limited in humans, chronic
* Correspondence: jcholewa@ coastal.edu
1
Department of Kinesiology, Recreation, and Sport Studies, Coastal Carolina
University, Conway, SC, USA
Full list of author information is available at the end of the article
© 2013 Cholewa et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Cholewa et al. Journal of the International Society of Sports Nutrition 2013, 10:39
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betaine supplementation has been shown to reduce adi-
pose mass and increase muscle mass in animals [7-9].
Greater improvements in body composition with betaine
supplementation were observed when pigs were given
extra pen space to move and exercise [9], suggesting that
betaine may exert the most influential effects on growth
under conditions of metabolic or nutritional stress. Be-
cause the subjects in Schwab et al. [6] and Del Favero
et al. [5] were instructed not to exercise, the absence of a
metabolic stressor may have compromised the effects of
betaine. Given the enhanced effects of movement in pigs
and the ineffectiveness reported in sedentary, non-
exercising humans, we hypothesize that the effects of
betaine on body composition, strength and power may be
most apparent when supplementation occurs over several
weeks accompanied by a resistance training program.
By donating a methyl group to transmethylate Hcy
back to methionine (Met), betaine increases Hcy meta-
bolism and the availability of the universal methyl donor,
S-adenosylmethion ine (SAM) [10]. We hypothesize beta-
ine supplementation may enhance protein synthesis and
thus improve body composition by reducing Hcy and
homocysteine thiolactone (HCTL). Hcy directly impairs
insulin signaling by reducing insulin receptor stubstrate-1
(IRS-1) activation and thus inhibiting Akt-phosphorylation
[11]. Moreover, excess dietary Met is metabolized to form
Hcy and both high dietary Met consumption and the
resultant increase in plasma Hcy contributes to elevated
HCTL [12]. A short (10 min) HCTL treatment inhibits
insulin signaling, including insulin-mediated mRNA ex-
pression and protein synthesis [13]. This suggests that
HCTL is more effective than Hcy in promoting insulin re-
sistance. Additionally, HCTL has been shown to modify
protein l ysine residues , which causes protein aggrega-
tion, and inactivates enzymes associated with protein
synthesis [14].
Concentrations of plasma Hcy or HCTL levels in
strength athletes have yet to be reported. Given that
transmethylation capacity is dependent upon plasma
folate and betaine [15] and because weight trainers regu-
larly consume excess Met and inadequate folate and
betaine [16], Hcy transmethylation may be impaired
resulting in excess HCTL generation. Thus, by decrea-
sing insulin receptor signaling [11], elevated HCTL in
weight lifters may compromise body composition directly
by inhibiting mRNA expression and protein synthesis.
In healthy adults the ingestion of 500 mg of betaine
decreased fasting plasma Hcy and attenuated Hcy rise
for 24 hr following a Met load [11], and betaine treat-
ment lowers HCTL in patients with genetically compro-
mised transmethylation capacities [12]; however, to date
there are no published repor ts investigating the effects
of betaine ingestion on HCTL in healthy subjects. We
hypothesize that by increasing transmethylation capacity
betaine supplementation reduces plasma Hcy and may
thus decrease HCTL generation, resulting in improved
insulin signaling and myofibril protein synthesis , and
ultimately enhancing muscle and strength gains. There-
fore, the purpose of this study was to investigate the
sub-chronic effects of betaine on strength, power, and
body composition during resistance training in experi-
enced streng th trained males. Additionally, urine HCTL
was measured to determine if betaine affects perform-
ance by reducing plasma HCTL. We hypothesized that
betaine supplementation would improve strength, verti-
cal jump, limb CSA, and body composition between the
1
st
week and 6
th
week over placebo. We also hypothe-
sized that betaine supplementation would reduce urinary
HCTL over the course of 6 weeks.
Methods
Experimental design
To investigate the effects of betaine supplementation, sub-
jects were matched, and randomly assigned to a treatment
or placebo group in a double-blinded study. Subjects
underwent 6 weeks of supplementation with either betaine
or placebo administered in identical gelatin capsules.
Before and after the treatment period skin fold and girth
measurements were taken, and subjects completed a
strength testing protocol. Additionally, urine was collected
prior to treatment and at 2 week intervals thereafter.
Subjects
Twenty three experienced recreationally strength trained
males (weight: 86.8 ± 9.1 kg; training experience : 4.8 ±
2.3 months; BF%: 16 .9 ± 8%) between the ages of 18 and
35 were recruited divided into two groups based on
training experience (6 month intervals) and body fat
percentage (2 percentage point intervals starting at 6%),
and randomly assigned to receive either the treatment
(n = 11) or placebo (n = 12). Medi cal histories were
obtained to exclude medical, musculoskeletal, and endo-
crine disorders, concurrent nutritional supplementation,
and anabolic drugs. Additionally, subjects must have
met the inclusion criteria to be classified as experienced
in resistance training [17]: previous consecutive resistance
training equal to or greater than 24 months; a frequency
of at least 3 resistance training sessions per week; at least
24 months experience in the back squat and bench press;
and the ability to bench press a load equal to body weight
and back squat at least 1.25 fold that of body weight. All
subjects signed an informed consent form following verbal
and written explanation of benefits and potential risks
associated with participating in the study.
Experimental controls
Subjects were required to complete a 3-day food diary,
and were instructed to consume a similar quantity/
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quality of foods throughout the study in order avoid
changes in nutritional status. Subjects were also required
to perform all prescri bed resistance training sessions,
complete and submit training logs to the primary inves -
tigator on a weekly basis, and abstain from performing
other structured exercise programs throughout the dur-
ation of the study. Subjects were required to render
urine upon waking following an overnight fast. Limb
girth, skin fold, strength, and power testing was carried
out at the same time of day within 2 days prior to and
immediately following the 6 week trial period. Prior to
all exercise test s, subjects wer e familiarized with the
assessment protocols. All methods and procedures were
approved by the Institutional Review Board of Springfield
College prior to data collection.
Procedures
All testing was conducted at the Springfield College
Human Performance Laboratory (HPL). Subjects were
required to report to the HPL on two separate occasions
(pre-treatment and post treatment) where height, nude
body mass, skin fold, anthropometric measurements,
and maximal strength testing was performed.
Body fat
Lange skin fold calipers (Cambridge Scientific Industries
Inc: Cambridge, MD) were calibrated before testing each
subject, and measurements were perform ed by the pri-
mary investigator to eliminate inter-rater variability. Skin
folds (mm) were measured on the right side of the body
in the following rotation: sub-scapular (X
1
), abdominal
(X
2
), triceps brachii (X
3
), and chest at the mix-auxiliary
line (X
4
). Body density (BD) was estimated via the follow-
ing equation [18]: BD = 1.03316 - .00164X
1
+.0041H -
.00144X
2
- .00069X
3
+ .00062X
4
, and then used to estimate
BF % [19]: BF % = [(4.57 / BD) – 4.142] × 100. Lean body
mass (LBM) and fat ma ss (FM) were then calculated
fromtheBF%andbodyweight.
Cross sectional area
A 6-week trial period was chosen to allow for detectable
changes in muscle CSA to occur. Changes in limb
muscle mass have been demonstrated to be detectable
via CSA measurements after four weeks of training and
continue to increase week to week [20]. Limb muscle
volume was assessed by evaluating differences in CSA
via the Moritani and DeVries (MD) method [21]. The
MD method is both sensitive (SEE = 3.25 cm
2
) and
highly correlated (r = .98) to computed tomography, the
gold standard of CSA measurement [22].
Girth and skin fold measurements were performed on
the right limbs to determine CSA via the MD method.
Cross sectional area of the arm was determined at the
midpoint between the humeral greater tuberosity and
lateral epicondyle, whereas CSA of the thigh wa s de-
termined at the midpoint of the distance between the
greater trochanter and lateral epicondyle of the femur.
Skin fold measurement s were performed three times
at the four quadrants of the limb at the location
where the circumference wa s measured. Cross se c-
tional area was calculated via the following equation
[21]: CSA ¼ π
C
2π
−
∑
4
i¼1
fi
4
,whereC = limb circumference
and ∑
4
i¼1
fi = sum of skin folds. All measurements were
performed by the primary investigator to eliminate inter-
rater variability. Distances from the proximal boney land
mark (humeral greater tuberosity and greater trochanter)
where measurements were performed were recorded and
used again for post treatment measuring to minimize
intra-rater variability.
Strength and power testing
All strength and power testing was conducted under the
supervision of a National Strength and Conditioning
Association (N SCA) Certified Strength and Conditioning
Specialist. Power was assessed via vertical jump using
the Just Jump! Mat (Probotics Inc.: Huntsville, AL).
Maximal strength was assessed with the free weight
bench press and back squat. The heaviest resistance
lifted in each exercise was considered the 1 RM. The
bench press and back squat were chosen for strength
assessment because: they are common exercises perfor-
med by weight lifters and the standardized strength
training program in this study utilized the two exercises.
Additionally, 1 RM testing has been shown to be a
reliable (ICC = .96) [17] measure to assess changes in
muscle strength following an exercise intervention.
All subjects completed a standardized dynamic warm
up prior to performance testing. Following a 5-min rest
subjects performed 3 trials of counter-movement vertical
jumps separated by a 3-min rest. Vertical jumps were
measured in inches on the Just Jump! mat. Subjects were
instructed to perform a rapid lower body eccentric con-
traction followed immediately by a maximal intensity
concentric contraction. Subjects were instructed to jump
straight up and minimize any in-air hip flexion. The best
of the three trials was recorded as vertical jump height.
Subjects were then given a 3-min rest prior to the
strength specific warm ups. Subjects performed three
sets of four repetitions with a progressively heavier load,
three sets of one repetition with a progressively heavier
load, and then a 3 min rest prior to attempting the first
1 RM. The first load used was 90% of the subject’s most
recent 1 RM or predicted from the subject’s most recent
RM [23]: 1-RM = 100 * rep wt / (101.3 – 2.67123 * reps).
Loads were inc reased by 5 – 10% and 10 – 20% for
bench press and squat, respectively, and then the 1 RM
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was determined in fewer than 5 set s with a rest inte rval
of 3–5 min between sets. There were no significant
differences in attempts between pre- and post-testing
(3.4 ± .82, p = .71). The bench press 1 RM was tested
first, and then a rest interval of at least 10 min was
provided prior to determining the back squat 1 RM.
Homocysteine thiolactone
HCTL is a toxic metabolite in humans and renal excre-
tion serves as the primary method of HCTL elimination
[14]. Urinary concentrations of HCTL are 100 fold
greater than those found in the plasma [24]. Urine was
rendered upon waking following an overnight fa st
prior to treatment administration (ba seline) and at the
end of week 2, 4 and 6 throughout the study. The u rine
samples were collected by the primary investigator on
the same d ay that urine wa s rendered and stored in
1-mL aliquot s at −80°C prior to being sent for analysis.
Urine was analyze d for HCTL via the cation-exchange
high pressure liquid chromatography (HPLC) at the
Institute of Bioorganic Chemistry, Polish Academy of Sci-
ences, Poznan, Dept. of Biochemistry and Biotechnology,
Life Sciences University, Poznan, Poland, as described
Jakubowski et al. [24-26]. The cation-exchange HPLC is
highly sensitive with a 0.36 nmol/L detection limit [24].
Treatments
Treatment s were administered double blind and consis-
ted of either a placebo (flour) or betaine (DuPont Nutrition
& Health: Tarrytown, NY). The blind was not removed
until all data had been collected. The primary investigator
filled identical, unmarked gelatin capsules with either
0.42 g white flour or 0.42 g betaine. Subject s consumed
three capsules (1.25 g) twice per day yielding an absolute
total of 2.5 g betaine. This dosage was chosen because:
betaineissafeatadietaryintakeof9– 12 g/day [1];
2.5 - 5 g beta ine has been shown to significantly elevate
plasma betaine [6]; 2.5 g positively affects strength
Table 1 Non-linear 6 week resistance training program
Monday Micro cycle 1 Micro cycle 2 Micro cycle 3
Exercise Week 1 - 2 Week 3 - 4 Week 5 - 6
Barbell Bench Press 3 × (12–15) / 90 sec 4 × (4–6) / 3 min 3 × (8–10) / 2 min
Barbell Incline Press 3 × (12–15) / 90 sec 4 × (4–6) / 3 min 3 × (8–10) / 2 min
Bent Over Barbell Row 3 × (12–15) / 90 sec 4 × (4–6) / 3 min 3 × (8–10) / 2 min
Seated Cable Row 3 × (12–15) / 90 sec 4 × (4–6) / 3 min 3 × (8–10) / 2 min
Biceps Barbell Curl 3 × (12–15) / 90 sec 4 × (4–6) / 3 min 3 × (8–10) / 2 min
Lying Triceps Extension 3 × (12–15) / 90 sec 4 × (4–6) / 3 min 3 × (8–10) / 2 min
Tuesday Micro cycle 1 Micro cycle 2 Micro cycle 3
Exercise Week 1 - 2 Week 3 - 4 Week 5 - 6
Free Weight Back Squat 3 × (8–10) / 2 min 3 × (12–15) / 90 sec 4 × (4–6) / 3 min
Barbell Rumanian Dead Lift 3 × (8–10) / 2 min 3 × (12–15) / 90 sec 4 × (4–6) / 3 min
Leg Extension Machine 3 × (8–10) / 2 min 3 × (12–15) / 90 sec 4 × (4–6) / 3 min
Abdominal Crunches 3 × (20–30) / 1 min 3 × (20–30) / 1 min 3 × (20–30) / 1 min
Thursday Micro cycle 1 Micro cycle 2 Micro cycle 3
Exercise Week 1 - 2 Week 3 - 4 Week 5 - 6
Barbell Military Press 3 × (12–15) / 90 sec 4 × (4–6) / 3 min 3 × (8–10) / 2 min
Wide Grip Front Lat Pull Down 3 × (12–15) / 90 sec 4 × (4–6) / 3 min 3 × (8–10) / 2 min
Dumbbell Row 3 × (12–15) / 90 sec 4 × (4–6) / 3 min 3 × (8–10) / 2 min
Dumbbell Lateral Shoulder Raise 3 × (12–15) / 90 sec 4 × (4
–6) / 3 min 3 × (8–10) / 2 min
Alternating Curls with Dumbbells 3 × (12–15) / 90 sec 4 × (4–6) / 3 min 3 × (8–10) / 2 min
Triceps Extension with Cables 3 × (12–15) / 90 sec 4 × (4–6) / 3 min 3 × (8–10) / 2 min
Friday Micro cycle 1 Micro cycle 2 Micro cycle 3
Exercise Week 1 - 2 Week 3 - 4 Week 5 - 6
Standard Dead Lift 3 × (8–10) / 2 min 3 × (12–15) / 90 sec 4 × (4–6) / 3 min
Barbell Split Squat 3 × (8–10) / 2 min 3 × (12–15) / 90 sec 4 × (4–6) / 3 min
Prone Leg Curl Machine 3 × (8–10) / 2 min 3 × (12–15) / 90 sec 4 × (4–6) / 3 min
Hanging Leg Raises 3 × (20–30) / 1 min 3 × (20–30) / 1 min 3 × (20–30) / 1 min
Note: Volume is prescribed as: Sets × (Rep Range) / Rest Periods.
Cholewa et al. Journal of the International Society of Sports Nutrition 2013, 10:39 Page 4 of 12
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performance [2,4]; and the average relative dosage (34.8
mg/kg-LBM) in the present study is similar to the average
relative dosage (36.3 mg/kg-LBM) reported previously to
improve performance [3]. Subjects were provided with 42
capsules per week and were required to log and submit
consumption times in accordance with the treatment
protocol.
Training program
Subjects assigned to the betaine and placebo groups
performed the same exercises, sets and repetitions du-
ring the six week investigation. A non-line ar periodiza-
tion (NLP) training program consisting of three 2-week
micro-cycles was prescribed because NLP has been shown
to produce larger increases in strength [27] and muscle
volume [28] compared to linear or non-periodized pro-
grams. Upper body training was performed on Monday
and Thursday, whereas lower body training took place
on Tuesday and Friday. The prescription for exercises,
training days, loads, rest period, warm ups, and resistance
progression can be found in Table 1.
The training sessions were not monitored; however, sub-
jects were required to submit training logs to the primar y
investigator on a biweekly basis (at the conclusion of each
micro-cycle). Training volume was calculated as the sum of
the load lifted multiplied by the number of repetitions
performed during each week for the bench press and back
squat, respectively. Work capacity for bench press and
back squat was assessed by comparing percent improve-
ment in training volume for each micro-cycle (week 1 vs.
week 2; week 3 vs. week 4; week 5 vs. week 6).
Statistical analysis
An independent samples t-test was used to examine
differences between groups for pre-trial BF % and
training experience. A 2 × 5 Mixed Factorial ANOVA
with Repeated Measures was used to determine the dif-
ference between groups (placebo and betaine) and time
for changes in urinary HCTL from baseline and week to
week. Two 2 × 6 Mixed Factorial ANOVA with Repeated
Measures were used to determine differences between
groups and time for bench press and back squat work
capacity at each training micro-cycle. If significant
interactions were found, percent improvements at each
micro-cycle was calculated and compared between
groups with an independent samples t-test. Eight 2 × 2
Mixed Factorial ANOVAs with Repeated Measures were
used to determine differences in arm CSA, thigh CSA,
BF %, LBM, FM, vertical jump, bench press 1 RM, and back
squat 1 RM between groups and time (pre- vs. post-trial).
All statistical analyses were analyzed using Statistical Pack-
age for the Social Sciences (SPSS v. 19, IBM) and the alpha
levelwassetat.05.
0
5
10
15
20
25
30
Week 2-1 Week 4-3 Week 6-5
Percent Change
Bench Press Training Volume
Placebo
Betaine
∗
∗
∗
Δ Δ Δ
Figure 1 Percent change in bench press volume for placebo (n = 12) and betaine (n = 11) for 3 training micro-cycles. Note: * = Significantly
(p < .05) different than placebo.
Table 2 Changes in bench press training volume (kg) for
placebo (n = 12) and betaine (n = 11) between three
micro-cycles
Pre Post Δ P
Micro Cycle 1
Betaine 2736 ± 463 2953 ± 500 216 ± 39 .01
Placebo 3154 ± 553 3170 ± 555 15 ± 70 .44
Micro Cycle 2
Betaine 1755 ± 296 1858 ± 315 103 ± 25 .30
Placebo 2320 ± 406 2903 ± 672 583 ± 288 .01
Micro Cycle 3
Betaine 2160 ± 365 2520 ± 427 360 ± 101 .01
Placebo 2481 ± 435 2779 ± 487 298 ± 62 .01
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Results
All values are presented as means ± standard deviations.
A significant interaction (p = .001) betw een group and
time existed for bench press work capacity (Figure 1).
Bench press training volume increa sed with placebo at
micro-cycles 2 and 3, and for betaine at micro-cycles 1
and 3 (Table 2). Post hoc analysis revealed the betaine
group improved significantly more than placebo at
micro-cycle one (7.89 ± 2.65% vs. 0.49 ± 1.69%, p = .001)
and three (16.67 ± 1.51% vs. 12.00 ± 4.21%, p = .05);
however, the percent improvement for placebo was
significantly greater than betaine at micro-cycle two
(19.2 ± 11.2% vs. 5.9 ± 1.4%, p = .001).
A significant main effect (p = .001) of time existed for
squat work capacity. Both groups increased squat vol-
ume at the se cond week of each training micro-cycle
(Figure 2 & Table 3); however, post-hoc analysis revealed
the percent improvement in squat training volume was
significantly greater with placebo than betaine at micro-
cycle one (14.3 ± 3.8% vs. 9.5 ± 0.8%, p = .001), and
significantly greater with betaine than placebo at micro-
cycle three (22.2 ± 1.3% vs. 10.7 ± 2.5%, p = .001). There
were no differences (p = .68) between groups for percent
improvement at micro-cycle two.
No significant (p = .70) main effect or interaction
existed between group and time for thigh CSA (Table 4).
A significant (p = .03) interaction was found between
groups and time for arm CSA (Figure 3). Arm CSA
increased significantly post-trial vs. pre-trial with betaine
but not placebo (Table 4).
All body composition data can be found in Table 5.
Significant interactions between group and time were
found for BF% (p =.007),LBM (p =.03), andFM (p =.01).
BF% and FM both decreased significantly post-trial vs.
pre-trial with betaine but not placebo (Figures 4, 5). Post-
trial LBM increased significantly over pre-trial with beta-
ine but not placebo.
Vertical jump, bench press 1RM and back squat 1Rm
data can be found in Table 6. An interaction trend (p = .07)
was found for vertical jump. Vertical jump decreased with
placebo and increased in betaine. No significant (p = .99)
interaction or main effect (p = .12) existed between group
and time for bench press. A significant (p = .001) main
effect for time w a s found for back squat 1 R M. Mean
post-trial back squat 1 R M was significantly greater
than pre-trial squat 1 RM; however, no significant inter-
action (p = .18) existed between group and time.
There was a trend (p = .06) for greater baseline HCTL
concentrations in betaine. A significant (p = .002) inter-
action between group and time was found for urinary
HCTL. The change in urinary HCTL with placebo was
significantly greater than that of betaine between ba se-
line and week 2, and baseline and week 4, respectively
(Figure 6 & Table 7). No significant changes in HCTL
were found for either group when comparing the change
between week 2 and 4 or week 4 and week 6; howe ver,
0
5
10
15
20
25
30
Week 2-1 Week 4-3 Week 6-5
Percent Change
Back Squat Training Volume
Placebo
Betaine
∗
∗
Δ Δ Δ
Figure 2 Percent change in back squat volume for placebo (n = 12) and betaine (n = 11) for 3 training micro-cycles. Note: * = Significantly
(p < .05) different than placebo.
Table 3 Changes in back squat training volume (kg) for
placebo (n = 12) and betaine (n = 11) between three
micro-cycles
Pre Post Δ P
Micro Cycle 1
Betaine 2760 ± 482 3022 ± 527 262 ± 43 .01
Placebo 3003 ± 695 3364 ± 779 360 ± 84 .01
Micro Cycle 2
Betaine 3736 ± 652 4084 ± 712 347 ± 76 .01
Placebo 4015 ± 930 4444 ± 1030 428 ± 159 .01
Micro Cycle 3
Betaine 2056 ± 357 2541 ± 444 484 ± 91 .01
Placebo 2350 ± 545 2655 ± 633 305 ± 85 .01
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a main effect of time wa s found when comparing week
6 to week 4.
Discussion
We hypothesized body composition would improve with
6 weeks of betaine supplementation. This hypothesis
was supported by sign ificant increases in lea n mass, and
decreases in fat mass and body fat percentage with beta-
ine compared to placebo. Increases in arm CSA were
found to be greater with betaine than placebo; however,
thigh CSA did not increase in either group. We also
expected strength and power performance to improve
with betaine supplementation. While back squat 1 RM
increased for both groups, there were no differences in
improvement between betaine and placebo. There was a
trend (p = .07) for greater vertical jump po wer with
betaine versus placebo, however there were no increases
in bench press 1 RM.
The improvements in lean mass, fat mass and body fat
percentage with betaine supplementation contrast previ-
ous investigations [5,6]. Differences in methodology may
explain these discrepancies: subject s in the previous
studies were both sedentary and instructed not to
exercise, whereas the subje cts in the present study were
currently training and given a structured exercise pro-
gram. Betaine has been suggested to act as a nutrient
partitioner and thereby accelerate lean mass gains in
pigs. By increa sing Hcy transmethylation, betaine spares
Met, allows for more efficient use of dietary protein, and
increases nitrogen retention [7]. Due to the inclusion of
resistance training in this study but not previous studies
[5,6], the demand for Met in the initiation of translation
in protein synthesis was likely elevated, thereby leading
to a greater utilization of elevated Met, and thus im-
provements in lean mass. Therefore, the results from the
present study lend support to the hypothesis that the
action of betaine to improve body composition in
humans may be most effective when accompanied by
exercise.
The increase in arm CSA in the betaine group com-
pared to placebo was accompanied by an improvement
in bench press work capacity. The greatest improve-
ments in volume over placebo occurred during the first
and third training micro-cycles, where subjects were
instructed to perform 3 sets of 12–15 repetitions with 90
sec rest periods and 3 sets of 8–10 repetitions with 120
sec rest periods, respectively. Given the relationship
between training volume and hypertrophy [29], betaine
may have positively impacted muscle growth by pro-
moting a greater training load over a series of subsequent
workouts.
The improvements in bench press work capacity differ
from previous studies where betaine did not improve
single-set repetitions to fatigue at 75% [3] or 3 sets of
repetitions to fatigue at 85% 1 RM [2]. In contrast, beta-
ine improved work capacity for 10 sets of repetitions to
fatigue at 50% 1 RM [4]. Given improved work capacity
with higher volume resistance training prescriptions,
and the lack of improvement during micro-cycle 2 which
imposed less of a metabolic demand (4 sets of 4–6
0
10
20
30
40
50
60
Placebo Betaine
Arm CSA (m
2
)
ARM Cross Sectional Area
Pre-Treatment
Post-Treatment
∗
Figure 3 Bar graph for arm cross sectional area (cm
2
) for placebo (n = 12) and betaine (n = 11) for pre- and post-treatment.
Note: * = Significantly (p < .05) different than pre-treatment.
Table 4 Changes in thigh and arm cross sectional area
(cm
3
) for placebo (n = 12) and betaine (n = 11) between
pre- and post-treatment
Pre Post Δ P
Thigh CSA
Betaine 85.0 ± 12.2 87.7 ± 12.2 2.7 ± 4.2 .254
Placebo 87.6 ± 17.7 89.0 ± 13.9 2.3 ± 10 .254
Arm CSA
Betaine 49.5 ± 8.7 54.1 ± 6.6 4.6 ± 4.3 .01
Placebo 53.4 ± 10.2 53.5 ± 11.2 -.1 ± 5 .98
Cholewa et al. Journal of the International Society of Sports Nutrition 2013, 10:39 Page 7 of 12
http://www.jissn.com/content/10/1/39
repetitions with 3 min rest), it is likely that betaine poses
the most ergogenic potential in resistance training exer-
cise protocols that impose higher metabolic demands.
Betaine is actively taken up by skeletal muscle during pe-
riods of stress, and may be ergogenic as an osmolyte by
protecting sensitive metabolic pathways against cellular
hypertonicity such as protein turnover, amino acid and
ammonia metabolism, pH regulation, and gene expres-
sion [30]. Specifically, betaine maintains cellular hydra-
tion to protect myosin ATPase and myosin heavy chain
proteins against denaturation by urea [31]. Moreover,
the affinity of troponin for Ca
2+
,
and thus force pro-
duction, is negatively affected by reductions in protein
hydration [32].
Contrary to the changes in arm CSA, no differen ces in
leg CSA were found between groups. Similar results
have been reported in animal studies investigating the
effects of betaine supplementation on carcass cuts where
betaine supplementation improved shoulder and butt,
but not ham meat yield [9]. Additionally, changes in
upper body muscle thickness occur at a greater
magnitude and earlier than do the lower extremities
[33]. Therefore, it is possible that changes in thigh CSA
may have occurred with a longer study period.
Although the back squat requires recruitment of the
quadriceps femoris, it also has a high gluteal/hip re-
quirement. Increases in muscle mass may have occurred
predominantly in the gluteals as seen in animal studies,
or the adaptations leading to greater back squat volume
and 1 RM occurred sep arately from increased muscle
CSA. Back squat work capacity increased for each group
at each training micro-cycle; however, the betaine group
improved nearly two-fold compared to placebo during
micro-cycle three (4 sets of 4–6 repetitions with 3 min
rest) which posed a higher neural and lower metabolic
demand than the previous micro-cycles. These impro-
vements in back squat work capacity contrasts previous
results [34] whereby betaine did not improve mean or
peak isokinetic power during 5 sets of 6 repetitions at
80% peak force. The improvements in work capacity at
micro-cycle three but not micro-cycle one or two also
contradict our hypothesis that betaine may be most
ergogenic when combined with exercise protocols pro-
ducing higher levels of metabolic stress. Given the
improvement in bench press work capacity that also
occurred at micro-cycle three but not two, and the
lack of improvement with only 2 weeks of sup-
plementation [2,4], it may also be that the effects of
increased intramuscular betaine manifest over a longer
period of time, and therefore require at least a 4–6week
ingestion period.
There were no differences between groups for back
squat 1 RM improvements , and despite increases in
bench press training volume with betaine, bench press 1
RM did not improve. This contrasts previous reports [2],
and may be partially explained by difference in subject
training status. Lee et al. employed recreationally trained
0
5
10
15
20
25
Placebo Betaine
BF %
Body Fat Percentage
Pre-treatment
Post-treatment
*
Figure 4 Bar graph for body fat percentage for placebo (n = 12) and betaine (n = 11) for pre- and post-treatment. Note: Significantly
(p < .05) different than pre-treatment.
Table 5 Changes in body composition for placebo (n = 12)
and betaine (n = 11) for pre- and post-treatment
Pre Post Δ P
Body Fat (%)
Betaine 17.5 ± 8.3 14.3 ± 5.7 −3.2 ± 2.5 .01
Placebo 16.4 ± 8.1 16.6 ± 8.2 0.2 ± 2.7 .82
Lean Body Mass (kg)
Betaine 69.5 ± 8.8 71.2 ± 7.9 2.4 ± 2.6 .01
Placebo 74.2 ± 9.1 74.5 ± 9.4 0.3 ± 2.6 .68
Fat Mass (kg)
Betaine 15.0 ± 7.9 12.1 ± 5.4 −2.9 ± 2.0 .01
Placebo 14.8 ± 8.0 15.1 ± 8.5 0.3 ± 2.3 .68
Cholewa et al. Journal of the International Society of Sports Nutrition 2013, 10:39 Page 8 of 12
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subjects, whereas subjects in the present study averaged
4.8 years of training experience. The ability to make
large performance gains, termed the “window of adap-
tation” [35], decreases with training experience. The
“window of adaptation” was likely smaller for the sub-
jects in the present study, thus reducing the ability to
detect changes in strength. Finally, the primary aim of
this study was to evaluate the effects of betaine on
muscle growth; thus, the training program utilized was
selected because it provided the greatest stimulation for
hypertrophy. Given the high training status of the sub-
jects, a strength concentrated program (i.e.: 4–6 sets of
1–3 repetitions) may have been needed to induce further
improvements in bench press and back squat 1 RM with
betaine supplementation.
There was a trend (p = .07) toward an increased vertical
jump with betaine supplementation. The positive trend in
the present study and improvements reported by Lee et al.
[2] differs from the results reported by other researchers
where vertical jump did not increase with betaine [3,4].
Variances in training prescription may account for these
discrepancies. In Lee et al. and the present study subjects
were assigned standardized training between testing
sessions, whereas subjects in Hoffman et al. [4] and
Trepanowski et al. [3] were not. Because detections in
power improvements are compromised when power
movements are not a regular part of training [34], future
researchers should include exercises that train muscular
contractile velocity when investigating the effects of beta-
ine supplementation on power output.
We hypothesized that subjects would have high urinary
HCTL values due to reduced Hcy transmethylational cap-
acity; however, the results did not support this hypothesis.
The normal range for urinary HCTL is .011-.473 nmol/
mL [24]. Mean pretreatment HCTL was .028 nmol/mL
(± .02 nnmol/mL), which suggests that the subje ct s
began t he study with low HCTL levels. Betai ne sup-
plementation attenuated the rise in HCTL obser ved in
placebo at weeks 2 and 4, but did not appear to reduce
HCTL values. Many subject s moved from the campus
dormitories to live with their parents for the summer.
It is possible that subject s had acc ess to foods higher in
protein quality and richer in fats and cholesterol than
when living on campus, and this led to the increase in
HCTL. Increases in dietary fat and cholesterol have
been shown to increa s e pla sma Hcy [36] as 3 Hcy are
produced during the methylation of phosphatid yle-
thanolamine in very low density lipoprotein synthesis.
Thus, higher met hionine and fat intakes may have
increased Hcy generation, leading to h igher le vels of
HCTL. Given the ability of betaine to increase Hcy
transmethylation, it is possible that betaine supplemen-
tation attenuated the dietary induced rise in HC TL.
HCTL decreased in both groups between week 4 and
week 6, although there was a trend for a reduction in
HCTL when comparing week 6 to baseline with betaine
and not placebo. While subjects were instructed to
maintain the same diet throughout the study, many
Table 6 Changes in vertical jump (cm), Back squat 1RM
(kg), and Bench press 1RM (kg) for Placebo (n = 12) and
Betaine (n = 11) for pre- and post-treatment
Pre Post Δ P
Vertical Jump
Betaine 68.1 ± 8.4 68.8 ± 8.4 0.8 ± 3.3 .45
Placebo 65.5 ± 10.4 63.0 ± 9.9 −2.5 ± 4.0 .09
Bench Press
Betaine 118.2 ± 19.3 120.0 ± 20.3 1.8 ± 4.3 .20
Placebo 137.7 ± 25.0 140.0 ± 24.5 2.3 ± 6.0 .31
Back Squat
Betaine 148.6 ± 26.7 151.4 ± 26.4 2.7 ± 4.5
*
.09
Placebo 159.1 ± 38.8 164.5 ± 38.1 5.5 ± 4.0 .01
* Non Significant Time × Treatment Interaction: p = .18.
30
35
40
45
50
55
60
65
70
75
80
Placebo Betaine
LBM (kg)
Lean Body Mass
Pre-treatment
Post-treatment
*
Figure 5 Bar graph for lean body mass (kg) for placebo (n = 12) and betaine (n = 11) for pre- and post-treatment. Note: Significantly
(p < .05) different than pre-treatment.
Cholewa et al. Journal of the International Society of Sports Nutrition 2013, 10:39 Page 9 of 12
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foods rich in betaine and fola te come into season in June
including spinach (0.3 mg/cup folate) and collard greens
(0.2 mg/cup folate), and the consumption of two-three
servings of folate rich food per day will reduce Hcy by
20% [37]. Because the start of June corresponded with
week 4 of the study, it is possible that the consumption
of local greens and the resultant increa se in folate con-
sumption may have reduced HCTL values in week 6.
Based on the small differences in HCTL changes, betaine
supplementation may have impacted body composition via
other mechanisms. Betaine has been shown to elevate
plasma GH and IGF-1, and increase Akt phosphorylation
in human skeletal muscle [38]. In mice betaine improves
insulin sensitivity by restoring activation of IRS1 and
the subsequent phosphorylation of PI3K/Akt by 50-100%
in a concentration-dependent manner [39]. Thus, i t is
possible that by ele vating anabolic hormones and
enhancing downstream cellular signaling , be taine may
have improved muscle protein synthesis , thus leading
to an increase in lean mass. Finally, because betaine is a
powerful osomylte, the increases in lean ma ss may have
been due to cellular swelling without an appreciable
increase in myofibril protein accretion.
Limitations
The MD method for estimating muscle CSA presents a
potential limitation when interpreting the limb CSA re-
sults of the present study. The SEE for the MD method is
3.25 cm
2
. In the present study, the betaine group increased
arm CSA by 4.6 cm
2
compared to a 0.1 cm
2
decrease with
placebo. The difference in change for thigh CSA between
betaine and placebo was 2.7 and 1.4 cm
2
, respectively. It is
possible that a non-significant difference in arm CSA
change or a significant difference in thigh CSA change
may have been observed if CSA was measured differently.
Future studies examining the effects of betaine on muscle
CSA change should utilize an analysis with a lower SEE.
Caution should also be taken when interpreting the
HCTL results. The primary aim in the present study was
to determine the effectiveness of betaine supplemen-
tation to improve strength and body composition in
weight trained males. A secondary aim was investigate if
a relationship between changes in HCTL values and
body composition or performance existed. Because
improvements in strength were reported in previous
studies without controlling for micronutrients [2,4], sub-
jects were instructed to consume a similar quantity and
quality of foods throughout the study to control for
energy and protein intake. Because subject diets were
not analyzed for micronutrients, it is possible that die-
tary fluctuations in folate, betaine, or other B-vitamin
consumption occurred and influenced urinary HCTL.
Future studies should provide standard control meals
and/or analyze micronutrient intake to investigate
Table 7 Changes in urinary homocysteine thiolactone
(nmol/mL) for Placebo (n = 12) and Betaine (n = 11)
between baseline and three time intervals
Concentration Δ From baseline P
Baseline
Betaine .037 ± .024
*
NA NA
Placebo .019 ± .018 NA NA
Week 2
Betaine .038 ± .02 .001 ± .02 .95
Placebo .049 ± .03 .029 ± .01 .01
Week 4
Betaine .039 ± .01 .002 ± .01 .74
Placebo .048 ± .02 .029 ± .01 .01
Week 6
Betaine .027 ± .03 -.024 ± .03 .29
Placebo .026 ± .02 .011 ± .03 .48
* Not significantly different than placebo at baseline: p = .06.
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
HCTL µmol/mL
Δ Week 2-BL Δ Week 4-BL Δ Week 6-BL
Placebo
Betaine
*
*
Figure 6 Changes in urinary homocysteine thiolactone values for placebo (n = 12) and Betaine (n = 11) between baseline and three
time intervals. Note: * = Significantly (p < .05) different than betaine.
Cholewa et al. Journal of the International Society of Sports Nutrition 2013, 10:39 Page 10 of 12
http://www.jissn.com/content/10/1/39
clinical relationships between betaine supplementation
and HCTL.
Conclusions
In summary, the major findings of the present study are
that 6 weeks of betaine supplementation improved body
composition, muscle size, work capacity, attenuated a rise
in HCTL, tended to improve power, but not strength in
resistance trained men. Further work is warranted to con-
firm any role of HCTL on body composition compared to
other mechanisms like lipogenic enzymatic activity,
growth hormones, cellular signaling, or gene expression.
Betaine attenuated an increase in urinary HCTL; however,
because strength trained men in this study had low base-
line HCTL betaine likely affected body composition via
another mechanism. HCTL has been implicated in vascu-
lar disease [40], insulin resistance [13], diabetic retinop-
athy [41], seizures, and Alzheimer’s disease [42]. Thus,
future investigations are needed to evaluate the clinical
ability of betaine to reduce HCTL in at risk populations
with elevated Hcy.
Competing interests
DuPont Nutrition & Health (Tarrytown, NY) provided funding for this project.
SASC is employed by DuPont Nutrition & Health. All other authors declare
they have no competing interests. All authors involved collected, analyzed,
or interpreted results from this study. Publication of these findings should
not be viewed as endorsement by the editorial board of the Journal of
International Society of Sports Nutrition.
Authors’ contributions
JMC was the primary investigator, designed the study, obtained grant funds,
supervised subject recruitment, data acquisition, data specimen collection,
and manuscript preparation. MWR, RG, and HJ performed data specimen
analysis. JMC was primarily responsible for writing the manuscript. TM, RW,
SASC, and VP made substantial contributions to manuscript writing and
preparation. All authors read and approved the final manuscript.
Author details
1
Department of Kinesiology, Recreation, and Sport Studies, Coastal Carolina
University, Conway, SC, USA.
2
Department of Environmental Chemistry,
University of Lodz, Pomorska 163, 90-236, Lodz, Poland, USA.
3
Institute of
Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Department of
Biochemistry and Biotechnology, Life Sciences University, Poznan, NJ, Poland.
4
UNDMJ-New Jersey Medical School, Newark, NJ, USA.
5
Department of
Exercise Science and Sport Studies, Springfield College, Springfield, MA, USA.
6
DuPont Nutrition & Health, Tarrytown, NY, USA.
Received: 14 March 2013 Accepted: 9 August 2013
Published: 22 August 2013
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Cite this article as: Cholewa et al.: Effects of betaine on body
composition, performance, and homocysteine thiolactone. Journal of the
International Society of Sports Nutrition 2013 10:39.
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