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9
Ergogenic and Antioxidant Effects of Spirulina
Supplementation in Humans
MARIA KALAFATI
1,2
, ATHANASIOS Z. JAMURTAS
1,2
, MICHALIS G. NIKOLAIDIS
1,2
, VASSILIS PASCHALIS
1,2
,
ANASTASIOS A. THEODOROU
1,2
, GIORGOS K. SAKELLARIOU
1,2
, YIANNIS KOUTEDAKIS
1,2,3
,
and DIMITRIS KOURETAS
4
1
Institute of Human Performance and Rehabilitation, Center for Research and Technology – Thessaly, Trikala, GREECE;
2
Department of Physical Education and Sport Science, University of Thessaly, Trikala, GREECE;
3
School of Sport,
Performing Arts and Leisure, Wolverhampton University, Walshall, UNITED KINGDOM; and
4
Department of Biochemistry
& Biotechnology, University of Thessaly, Larissa, GREECE
ABSTRACT
KALAFATI, M., A. Z. JAMURTAS, M. G. NIKOLAIDIS, V. PASCHALIS, A. A. THEODOROU, G. K. SAKELLARIOU,
Y. KOUTEDAKIS, and D. KOURETAS. Ergogenic and Antioxidant Effects of Spirulina Supplementation in Humans. Med. Sci.
Sports Exerc., Vol. 42, No. 1, pp. 142–151, 2010. Purpose: Spirulina is a popular nutritional supplement that is accompanied by
claiMSS for antioxidant and performance-enhancing effects. Therefore, the aim of the present study was to examine the effect of
spirulina supplementation on (i) exercise performance, (ii) substrate metabolism, and (iii) blood redox status both at rest and after
exercise. Methods: Nine moderately trained males took part in a double-blind, placebo-controlled, counterbalanced crossover study.
Each subject received either spirulina (6 gId
j1
) or placebo for 4 wk. Each subject ran on a treadmill at an intensity corresponding to
70%–75% of their V
˙O
2max
for 2 h and then at 95% V
˙O
2max
to exhaustion. Exercise performance and respiratory quotient during
exercise were measured after both placebo and spirulina supplementation. Blood samples were drawn before, immediately after, and at
1, 24, and 48 h after exercise. Reduced glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG, thiobarbituric acid-reactive
substances (TBARS), protein carbonyls, catalase activity, and total antioxidant capacity (TAC) were determined. Results: Time to
fatigue after the 2-h run was significantly longer after spirulina supplementation (2.05 T0.68 vs 2.70 T0.79 min). Ingestion of spirulina
significantly decreased carbohydrate oxidation rate by 10.3% and increased fat oxidation rate by 10.9% during the 2-h run compared
with the placebo trial. GSH levels were higher after the spirulina supplementation compared with placebo at rest and 24 h after
exercise. TBARS levels increased after exercise after placebo but not after spirulina supplementation. Protein carbonyls, catalase, and
TAC levels increased similarly immediately after and 1 h after exercise in both groups. Conclusions: Spirulina supplementation
induced a significant increase in exercise performance, fat oxidation, and GSH concentration and attenuated the exercise-induced
increase in lipid peroxidation. Key Words: FREE RADICALS, REACTIVE OXYGEN SPECIES, REDOX STATUS, OXIDATIVE
STRESS, PHYSICAL ACTIVITY
Spirulina (Spirulina platensis) is a photosynthetic cy-
anobacterium that possesses biological activity and
is widely cultivated to produce nutritional supple-
ments (26). Spirulina is rich in essential amino acids and
fatty acids (palmitic acid, linoleic acid, and F-linolenic acid),
vitamin C, vitamin E, and selenium (26). Recently, attention
has been placed on the antioxidant potential of spirulina.
Indeed, many of the chemical components of spirulina,
such as phenolic compounds, tocopherols,
A
-carotenes, and
phycocyanins exhibit antioxidants properties (11). For in-
stance, it has been reported that spirulina supplementation
with ginseng decreased lipid peroxidation and increased the
levels of reduced glutathione (GSH), superoxide dismutase,
and glutathione peroxidase in the kidney of rats (21).
Exercise promotes the production of reactive oxygen and
nitrogen species (RONS). Growing evidence indicates that
RONS contribute to muscle fatigue (14). To protect against
exercise-induced oxidative damage, cells contain endoge-
nous cellular defense mechanisMSS to control the levels of
RONS (37). Furthermore, exogenous dietary antioxidants
interact with endogenous antioxidants and form a network of
cellular antioxidants (37). The fact that exercise-induced
RONS production can contribute to muscle fatigue (14) has
resulted in numerous investigations examining the effects of
different antioxidants (e.g., vitamin C or N-acetylcysteine)
on human redox status and exercise performance (e.g.,
[2,28]). However, comparatively few researchers have
studied the effect of foods rich in antioxidants on oxidative
stress provoked by exercise (34,45). Thus, the extent to
Address for correspondence: Athanasios Z. Jamurtas, Ph.D., Department of
Physical Education and Sports Sciences, University of Thessaly, Karies,
42100, Trikala, Greece; E-mail: ajamurt@pe.uth.gr.
Submitted for publication November 2008.
Accepted for publication April 2009.
0195-9131/10/4201-0142/0
MEDICINE & SCIENCE IN SPORTS & EXERCISE
!
Copyright "2009 by the American College of Sports Medicine
DOI: 10.1249/MSS.0b013e3181ac7a45
142
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9
which foods rich in antioxidants (such as spirulina) modify
the redox status responses induced by exercise is largely
unknown.
We found only one study that examined the effects of
spirulina on redox status and exercise performance (25).
However, the blood samples after spirulina supplementation
and exercise were compared with the resting blood samples
making it difficult to discern the spirulina effect. Nowadays,
spirulina is a very popular nutritional supplement for
humans and is accompanied by claiMSS for antioxidant
and performance-enhancing effects (12). These claiMSS are
extrapolated by the findings of in vitro and animal studies
(11,21) but have not been substantiated concerning humans.
Therefore, the aim of the present study was to examine the
effect of spirulina supplementation on (i) exercise perfor-
mance, (ii) substrate metabolism, and (iii) blood redox
status both at rest and after exercise.
MATERIALS AND METHODS
Subjects. Nine healthy moderately trained men (age =
23.3 T1.7 yr, height = 174.3 T1.7 cm, weight = 70.7 T
1.9 kg, body fat = 9.8 T1.3%, maximal oxygen consump-
tion (V
˙O
2max
) = 52.2 T1.8 mLIkg
j1
Imin
j1
) volunteered to
participate. The subjects were recreational runners and had
trained for at least 1 yr (3.4 T1.1 yr), at least two times per
week (3.1 T0.9 times per week), at least 45 min per session
(56 T10 min per session). All subjects were informed
thoroughly about the risks, the possible discomforts, and the
benefits of the study before signing a written informed
consent. All subjects completed a medical and supplemen-
tation history and physical activity questionnaire to deter-
mine eligibility. No subject was a smoker or taking
supplements or anti-inflammatory drugs. The procedures
were in accordance with the Helsinki Declaration of 1975
and approved by the institutional review board.
Baseline measurements. One to two weeks before
the first exercise trial, subjects visited the laboratory for
baseline measurements. Body mass was measured to the
nearest 0.5 kg with subjects lightly dressed and barefoot
(Beam Balance 710; Seca, Birmingham, United Kingdom)
and standing height was measured to the nearest 0.5 cm
(Stadiometer 208; Seca). Percentage body fat was calculated
from seven skinfold measurements using a Harpenden skin-
fold caliper (John Bull, British Indicators Ltd, St. Albans,
United Kingdom) according to published guidelines (4). To
establish that all subjects ran at similar exercise intensity,
V
˙O
2max
was determined using a treadmill test to exhaustion.
The protocol began at 10 kmIh
j1
and was increased by
1 km every 2 min until V
˙O
2max
was reached. V
˙O
2max
test
was terminated when three of the following four criteria
were met: (i) subject exhaustion, (ii) a G2 mLIkg
j1
Imin
j1
increase in V
˙O
2
with an increase in work rate, (iii) a
respiratory exchange ratio Q1.10, and (iv) an HR within 10
bpm of the theoretical maximum HR (220 jage).
Respiratory gas variables were measured using a metabolic
cart (Vmax29; SensorMedics, Yorba Linda, CA), which
was calibrated before each test using standard gases of
known concentration. Exercise HR was monitored by
telemetry (Tester S610
i
; Polar, Electro Oy, Finland).
Study design. A double-blind, placebo-controlled,
counterbalanced crossover design was used (i.e., half of
the subjects were given the spirulina first and the other half
were given the placebo and the reversed). Each subject
participated in four exercise trials (Fig. 1). In the first
exercise trial, subjects visited the laboratory 7–14 d after
FIGURE 1—Study design. Arrows indicate blood sampling.
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d
143
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9
V
˙O
2max
determination (between 08:00 and 10:00), where
they ran on a treadmill at an intensity corresponding to
70%–75% of their V
˙O
2max
for 2 h. After the 2-h run, the
speed of the treadmill was increased to elicit the 95%
V
˙O
2max
, and exercise was terminated at exhaustion (31).
Fatigue was considered to have occurred when the required
speed could not be maintained by the subject or when the
subject stopped voluntarily. The time to reach volitional
fatigue was recorded and used as an index of aerobic
performance. Expired gas samples were obtained every
10 min to ensure the prescribed exercise intensity and to
calculate the fat and carbohydrate oxidation rates. Water
(250 mL) was given to the volunteers every 20 min during
exercise. After the end of the initial exercise trial, each
subject consumed two capsules (1 g each) containing either
S. platensis manufactured by Algae AC (Serres, Greece) or
100% egg protein (placebo). The capsules were consumed
before meals three times per day for 4 wk. The daily dosage
of spirulina that was used (6 gId
j1
) was close to other
relevant human studies (7.5 [15] and 8 [25] gId
j1
). One day
after the end of the 4-wk supplementation period, subjects
came back to the laboratory to perform the second exercise
bout with identical conditions as the first exercise trial. A
2-wk washout period occurred between the second and the
third exercise trials to avoid possible carryover effects.
After the washout period, the subjects came back for a third
and fourth times, where the exercise conditions of the first
and second exercise trials were followed. The first and third
exercise trials were performed to ensure that the 2-wk
washout period was adequate to have similar physiological
and biochemical values before the two periods of supple-
mentation. We are aware of only one study that investi-
gated the effects of spirulina supplementation on humans
using a crossover design (6). In this study, a 2-wk washout
period was also used. In addition, taking into account the
short supplementation period used in the present study (i.e.,
4 wk), we considered that the 2-wk washout period would
be long enough for any effects of placebo or spirulina to
disappear.
The basic composition of dry spirulina is as follows: 63.3%
protein, 7.1% lipid, and 15.2% carbohydrate (50), 101 mg of
vitamin C (5), 15 mg of vitamin E, and 0.13 mg of selenium
per 100 g (50), as well as 43.6% palmitic acid, 17.2% lino-
leic acid, and 21.7% F-linolenic acid of total fatty acids (33).
Fat and carbohydrate oxidation. Fat and carbohy-
drate oxidation rates (gImin
j1
) were calculated indirectly
by monitoring the rate of O
2
consumption (LImin
j1
) and
CO
2
production (LImin
j1
) using the following stoichiomet-
ric equations (18), assuming that protein oxidation during
exercise was negligible:
fat oxidation = 1.695V
˙O
2
– 1.701V
˙CO
2
carbohydrate oxidation = 4.210V
˙CO
2
– 2.962V
˙O
2
Blood collection and handling. Blood samples were
drawn from a forearm vein at rest and after exercise
(immediately after exercise and at 1, 24, and 48 h after
exercise). Directly after taking the blood sample, 0.5 mL of
blood was placed in a tube containing EDTA for the
determination of hematocrit and hemoglobin. Whole-blood
lysate was produced by adding 5% trichloroacetic acid
(TCA) to whole blood (1:1 v/v) collected in EDTA tubes
for reduced GSH and oxidized glutathione (GSSG) analy-
sis. The whole-blood samples were centrifuged at 4000gfor
10 min at 4-C, and the supernatant was removed and
centrifuged again at 28,000gfor 5 min at 4-C. The clear
supernatant was collected in Eppendorf tubes and stored
at j80-C until GSH and GSSG determination. Another
portion of blood was collected in plain tubes, left on ice
for 20 min to clot, and centrifuged at 1500gfor 10 min at
4-C for serum separation. Serum was transferred in
Eppendorf tubes and was used for the determination of
creatine kinase, thiobarbituric acid-reactive substances
(TBARS), protein carbonyls, catalase, and total antioxidant
capacity (TAC). Serum samples were stored in multiple
aliquots at j80-C and were thawed only once before
analysis.
Assays. A slightly modified version of Reddy et al. (40)
was used to measure GSH, which is originally based on
Beutler et al. (7). Twenty microliters of whole blood treated
with TCA was mixed with 660 KL of 67 mM sodium
potassium phosphate (pH 8.0) and 330 KL of 1 mM 5,5-
dithiobis-2-nitrobenzoate (DTNB). The samples were incu-
bated in the dark at room temperature for 45 min, and the
absorbance was read at 412 nm. A standard curve was
constructed by using GSH as a standard at concentrations of
0, 0.25, 0.50, and 1 mM. GSSG was determined according
to Tietze (49). Two hundred and sixty microliters of whole
blood treated with TCA was neutralized up to pH 7.0–7.5
with NaOH. Four microliters of 2-vinyl pyridine was added,
and the samples were incubated for 2 h at room tempera-
ture. Five microliters of whole blood treated with TCA was
mixed with 600 KL of 143 mM sodium phosphate (6.3 mM
EDTA, pH 7.5), 100 KL of 3 mM nicotinamide dinucleo-
tide phosphate (NADPH), 100 KL of 10 mM DTNB, and
194 KL of distilled water. The samples were incubated for
10 min at room temperature. After the addition of 1 KL of
glutathione reductase, the change in absorbance at 412 nm
was read for 3 min. A standard curve was constructed by
using GSSG as a standard at concentrations of 0, 0.025,
0.050, and 0.100 mM. The GSH/GSSG ratio was calculated
for each subject, and the means of these ratios for each time
point are presented.
TBARS were measured according to Keles et al. (22). One
hundred microliters of serum was mixed with 500 KL of
35% TCA and 500 KL of Tris–HCl (200 mM, pH 7.4) and
incubated for 10 min at room temperature. One microliter of
2 M Na
2
SO
4
and 55 mM thiobarbituric acid solution was
added, and the samples were incubated at 95-C for 45 min.
The samples were cooled on ice for 5 min and were vortexed
after adding 1 mL of 70% TCA. Finally, the samples were
centrifuged at 15,000gfor 3 min, and the absorbance of the
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9
supernatant was read at 530 nm. A standard curve was con-
structed by using malondialdehyde as a standard at concen-
trations of 0, 1.25, 2.5, 5, and 10 KM.
Protein carbonyls were measured according to Patsoukis
et al. (36). In 50 KL of serum, 50 KL of 20% TCA was
added, incubated in the ice bath for 15 min, and centrifuged
at 15,000gfor 5 min at 4-C. The supernatant was discarded,
and 500 KL of 10 mM 2,4-dinitrophenylhydrazine (in 2.5N
HCl) for the sample, or 500 KL of 2.5N HCl for the blank,
was added to the pellet. The samples were incubated in
the dark at room temperature for 1 h, with intermittent
vortexing every 15 min, and were centrifuged at 15,000g
for 5 min at 4-C. The supernatant was discarded, and 1 mL
of 10% TCA was added, vortexed, and centrifuged at
15,000gfor 5 min at 4-C. The supernatant was discarded,
and 1 mL of ethanol–ethyl acetate (1:1 v/v) was added,
vortexed, and centrifuged at 15,000gfor 5 min at 4-C. The
washing step was repeated two more times. The supernatant
was discarded, and 1 mL of 5 M urea (pH 2.3) was added,
vortexed, and incubated at 37-C for 15 min. The samples
were centrifuged at 15,000gfor 3 min at 4-C, and the
absorbance was read at 375 nm. Protein carbonyls values
were obtained by using the extinction coefficient of 2,4-
dinitrophenylhydrazine (22 mMIcm
j1
).
Catalase activity was measured according to Aebi (1). In
20 KL of serum, 2975 KL of 67 mM sodium potassium
phosphate (pH 7.4) was added, and the samples were
incubated at 37-C for 10 min. Five microliters of 30%
hydrogen peroxide was added to the samples, and the
change in absorbance was immediately read at 240 nm for
1.5 min. Catalase activity was obtained by using the
extinction coefficient of hydrogen peroxide (43.6 MIcm
j1
).
TAC was measured according to Janaszewska and
Bartosz (17). For TAC, in 20 KL of serum, 480 KL of 10
mM sodium potassium phosphate (pH 7.4) and 500 KL of
0.1 mM 2,2-diphenyl-1 picrylhydrazyl (DPPH) were added
and incubated in the dark for 30 min at room temperature.
The samples were centrifuged for 3 min at 20,000g, and the
absorbance was read at 520 nm. TAC values were obtained
by calculating the number of DPPH molecules scavenged
per minute.
Serum creatine kinase was determined spectrophotomet-
rically using a commercially available kit (Spinreact, Sant
Esteve, Spain). Total protein in serum was assayed using a
Bradford reagent. Postexercise plasma volume changes
were computed based on hematocrit and hemoglobin.
Hematocrit was measured by microcentrifugation, and
hemoglobin was measured using a kit from Spinreact.
Each assay was performed in duplicates, except for GSSG,
which was performed in triplicates. The intra-assay
coefficient of variation for each measurement was as
follows: GSH 4.0%, GSSG 6.5%, TBARS 3.9%, protein
carbonyls 5.5%, catalase 6.7%, TAC 3.7%, and creatine
kinase 2.9%.
Dietary analysis. To factor the effect of the diet on the
outcome measures of the study and to establish that
participants had similar levels of macronutrient and anti-
oxidant intake during the period of data collection, they
were asked to record their diet for 3 d preceding their first
visit to the laboratory and to repeat this diet before their
next three visits to the laboratory. Each subject had been
provided with a written set of guidelines for monitoring
dietary consumption and a record sheet for recording food
intake. Diet records were analyzed using the nutritional
analysis system ScienceFit Diet 200A (ScienceFit, Athens,
Greece).
Statistical analysis. The distribution of all dependent
variables was examined by the Shapiro–Wilk test and was
found not to differ significantly from normal. First, to
ensure that the 2-wk washout period was adequate, the
data from the first and the third trials were analyzed
through two-way (trial !time) ANOVA with repeated
measures on time. Second, to evaluate the effects of
supplementation and exercise, the data from the second
and the fourth trials were analyzed through two-way
(group !time) ANOVA with repeated measures on time.
If a significant interaction was obtained, pairwise com-
parisons were performed through simple main effect
analysis. Differences in diet among trials or groups were
examined through one-way ANOVA. Aerobic perfor-
mance at the second and fourth exercise trials was
examined by paired t-test. Carbohydrate and lipid oxidation
rates during the 2-h run at the second and fourth exercise
trials were also examined by paired t-test. Statistical
significance was considered when PG0.05. The SPSS
version 15.0 was used for all analyses (SPSS, Inc., Chicago,
IL). Data are presented as mean TSEM.
RESULTS
Washout, compliance, and diet. The comparison of
the data from the first and the third trials revealed no
significant interaction and no significant main effect of trial
on any of the dependent variables measured. Therefore, the
2-wk washout period proved adequate to have similar
physiological and biochemical values before the two
periods of supplementation. Supplementation compliance
was 97.6% and 96.4% for placebo and spirulina, respec-
tively, as revealed by the counting of the capsules provided
upon return of the bottles. No adverse effects were reported
after spirulina supplementation. Dietary intake, assessed
during the 3-d period, showed no differences between
groups in any of the assessed variables (Table 1).
Exercise performance. The average exercise inten-
sity during the 2-h submaximal run for the placebo and
spirulina trials was 70.6 T2.4% and 71.0 T1.9 % of
V
˙O
2max
, respectively (P90.05). Time to fatigue after the
2-h run was significantly higher after spirulina supplemen-
tation (2.05 T0.68 vs 2.70 T0.79 min for the placebo and
spirulina groups, respectively, P= 0.048; Fig. 2). Time to
fatigue at 95% V
˙O
2max
was reproducible in preliminary
trials (coefficient of variance (CV) 6.2 T0.7%).
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Fat and carbohydrate oxidation. Supplementation
of spirulina significantly decreased carbohydrate oxidation
rate by 10.3% (P= 0.008) and increased fat oxidation rate
(P= 0.003) by 10.9% during the 2-h run compared with
the placebo trial (Fig. 3).
Plasma volume. Plasma volume did not change during
the 48-h postexercise period in both groups (P90.05);
nevertheless, the values were corrected for any nonsignifi-
cant plasma volume changes.
Creatine kinase. There was no significant main
effect of group or time !group interaction concerning
serum creatine kinase (Fig. 4). However, there was a
significant main effect of time (PG0.001), with creatine
kinase activity increasing 24 and 48 h after exercise in
both groups.
GSH status. There was no significant main effect of
time or group !time interaction concerning GSH
(Fig. 5A). However, there was a significant main effect of
group (P= 0.049), with GSH level being higher after the
spirulina supplementation at rest and 24 h after exercise.
There were no significant main effects or interactions for
GSSG and GSH/GSSG ratio (Figs. 5B and C).
TBARS and protein carbonyls. There was no signif-
icant main effect of group or time concerning serum
TBARS (Fig. 6A). However, there was a significant
group !time interaction (P= 0.007), with TBARS levels
increasing after exercise after placebo but not after spirulina
supplementation. There was no significant main effect of
group or time !group interaction concerning serum protein
carbonyls (Fig. 6B). However, there was a significant main
effect of time (PG0.001), with protein carbonyls levels
increasing immediately after and 1 h after exercise in both
groups.
Catalase and TAC. There was no significant main
effect of group or group !time interaction concerning se-
rum catalase (Fig. 7A). However, there was a significant
main effect of time (PG0.001), with catalase activity increas-
ing immediately after and 1 h after exercise in both groups.
There was no significant main effect of group or group !time
interaction concerning serum TAC (Fig. 7B). However, there
was a significant main effect of time (PG0.001), with TAC
increasing immediately after and 1 h after exercise in both
groups.
DISCUSSION
To our knowledge, this is the first attempt to examine the
effects of spirulina supplementation on exercise performance,
FIGURE 3—Oxidation rate in the placebo and spirulina trial during
the 2-h run (mean TSEM). *The carbohydrate and fat rates were
significantly different between the placebo and the spirulina trials
(PG0.05).
FIGURE 2—Exercise performance at 95% V
˙O
2max
in the placebo and
spirulina trial (mean TSEM). *Significantly different from the placebo
trial (PG0.05).
FIGURE 4—Creatine kinase (CK) activity in the placebo (open
rectangles) and spirulina exercise trials ( filled rectangles; mean T
SEM). *Significantly different from the resting value in the same trial
(PG0.05).
TABLE 1. Analysis of daily energy intake after placebo and spirulina supplementation
(mean TSEM).
Placebo Spirulina
Energy (kcal) 2537 T127 2421 T61
Carbohydrate (% energy) 44.0 T2.8 47.0 T1.7
Fat (% energy) 39.0 T3.1 37.1 T2.9
Protein (% energy) 17.0 T1.3 15.9 T1.6
Vitamin A (mg, RE) 999 T164 663 T177
Vitamin C (mg) 169 T18 162 T18
Vitamin E (mg,
>
-TE) 11.0 T1.4 11.5 T1.3
Selenium (Kg) 155 T11 123 T5
>
-TE,
>
-tocopherol equivalents; RE, retinol equivalents.
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9
substrate metabolism, and blood redox status at rest and after
exercise in humans. The results showed that spirulina
supplementation for 4 wk induced a significant increase in
exercise performance, fat oxidation, and glutathione concen-
tration as well as attenuated exercise-induced increases in lipid
peroxidation. This provides evidence that increased levels of
fat oxidation and GSH may contribute to enhanced exercise
performance.
Exercise performance and increased fat oxida-
tion rate. Probably the most interesting finding of the
present study is the increase in exercise performance after
spirulina supplementation. Despite the fact that the mecha-
nism behind the ergogenic effect of spirulina is difficult
to be identified, the most plausible explanation implicates
fat oxidation, the rate of which was found substantially
increased (15.8%) during the 2-h exercise trial in spirulina-
supplemented individuals. The maintenance of maximal
aerobic power output requires that carbohydrates are
oxidized as well as fats (15). Because carbohydrates come
from the glycogen stores, the time that maximal aerobic
power can be sustained depends on the amount of glycogen
stored initially (15). In fact, it was found that the time to
exhaustion when working at 75% of maximal aerobic
power (almost equal to 70% V
˙O
2max
that was used in the
present study) correlated with the initial muscle glycogen
concentration (15). Moreover, there is evidence that in-
creasing fat oxidation leads to sparing of glycogen (15);
thus, at least in principle, the increased fat oxidation could
have spared glycogen or glucose to allow high-intensity
exercise to be continued for a longer time.
We have no hint as to what biochemical mechanism may
have led to increased fat oxidation after spirulina supple-
mentation, partly because spirulina is a complex mixture of
substances with different properties. Potential control points
of fat oxidation include lipolysis in adipose tissue,
transportation of fatty acids via blood, transportation of
fatty acids to muscle, hydrolysis of myocellular triacylgly-
cerols, transportation of fatty acids to mitochondria, and
mitochondrial density (30). We know very little about
whether and how spirulina affects these processes. How-
ever, the high content of F-linolenic acid in spirulina
(21.7% of total fatty acids in dry spirulina [33]) may play
a role in mediating the reported effects on fat metabolism in
the present study. In fact, F-linolenic acid has been shown
to reduce body fat (47) and facilitate fatty acid A-oxidation
in the liver as judged by the increased activities of carnitine
palmitoyl-transferase (24,47), acyl-CoA oxidase (24), and
peroxisomal
A
-oxidation (47) in rats.
Exercise performance and increased GSH con-
centration. Except for the substrate-oriented explanation
depicted in the previous paragraphs, the increased concen-
tration of GSH may also explain to some extent the
increased performance detected after spirulina supplemen-
tation. Several studies provided convincing data to support
the view that cysteine is generally the limiting amino acid
for GSH synthesis in humans and in other animals (54).
FIGURE 5—GSH (A) and GSSG concentrations (B) as well as GSH/GSSG (C) ratio in the placebo (open rectangles) and spirulina exercise trials
(filled rectangles; mean TSEM). #Significantly different between placebo and spirulina trial at the same time point (PG0.05).
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Thus, increasing the supply of cysteine or its precursors
(e.g., N-acetylcysteine) via oral or intravenous administra-
tion enhances GSH synthesis (28,44). Because cysteine can
be generated from the catabolism of sulfur-containing
methionine via the transsulfuration pathway, dietary me-
thionine can replace cysteine to support GSH synthesis
in vivo (54).
Spirulina contains 0.45 g of cysteine and 1.25 g of me-
thionine per 100 g of dry spirulina (42). Given that subjects
of the present study received 6 g spirulina per day, they re-
ceived approximately 27 mg of cysteine and 75 mg methio-
nine per day from spirulina. An analysis of the amino acid
intake received by the subjects through their diet revealed
that the subjects consumed approximately 3417 mg of
cysteine and 6953 mg of methionine every day. This
translates to a 0.79% increase in cysteine and 1.08% increase
in methionine daily consumption solely from spirulina. It is
possible that this small (but stable and dispersed throughout a
day) administration of cysteine and methionine for the 4 wk
of the supplementation period led to the increased concen-
tration of GSH. Indeed, increased GSH concentration after
spirulina supplementation has been reported in studies of the
kidney (21,23), liver (21,38), lung (52), heart (48,52), and
blood of rats (48).
Another potential mechanism that may have led to the
increased levels of GSH after spirulina supplementation is
the increased content of vitamins C and E in spirulina (54).
In fact, vitamin C, vitamin E, and GSH undergo redox
cycling in vivo, and there seeMSS to be a significant
interrelationship among the three molecules in this cycling
(54). Supporting this fact, several studies have indicated
increased levels of GSH after supplementation with vita-
mins C and E (54).
GSH levels seem to be important in controlling the levels
of RONS and muscle function (14). N-Acetylcysteine, a
drug that supports GSH synthesis, has been consistently
shown to delay muscle and whole-body fatigue. In humans,
N-acetylcysteine administration improved performance of
limb muscles (41) and diaphragm (51) during increased
contractile activity protocols and extended time to failure
during whole-body exercise (27,28). Overall, the role that
RONS play in fatigue is still unclear (14), and conse-
quently, the potential mechanisMSS through which the
increased levels of GSH may have affected whole-body
endurance in the present study are difficult to be predicted.
Effect of spirulina supplementation on redox
status at rest. The only difference found in the present
study regarding the redox status at rest was the higher
concentration of GSH detected in spirulina-supplemented
individuals. Despite a fair number of studies conducted in
animals (e.g., [21,23,38,48,52]), we found only two studies
that addressed the effects of spirulina supplementation on
FIGURE 6—TBARS (A) and protein carbonyl (B) concentrations in
the placebo (open rectangles) and spirulina exercise trials (filled
rectangles; mean TSEM). *Significantly different from the resting
value in the same trial (PG0.05). #Significantly different between
placebo and spirulina trial at the same time point (PG0.05).
FIGURE 7—Catalase activity (A) and total antioxidant capacity (B) in
the placebo (open rectangles) and spirulina exercise trials ( filled
rectangles; mean TSEM). *Significantly different from the resting
value in the same trial (PG0.05).
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9
redox status in humans (35,43). The two studies measured
several indices of redox status in blood and reported
contradictory results. For example, Park et al. (35) reported
decreased levels of lipid peroxidation, whereas Shyam et al.
(43) reported no change in lipid peroxidation after spirulina
supplementation.
Effect of spirulina supplementation on redox
status after exercise. TBARS was the only biochem-
ical variable that a significant group !time interaction
was detected, with TBARS levels increasing after exercise
after placebo but not after spirulina supplementation. The
main probable mechanism through which exercise in-
creased lipid peroxidation after its cessation is the
increased susceptibility to peroxidation of unsaturated
fatty acids (16) because exercise markedly increases the
concentration and unsaturation degree of nonesterified
fatty acids in blood (32). The higher levels of GSH can
partially explain the absence of an increase in lipid
peroxidation after exercise in the spirulina-supplemented
individuals. GSH can effectively scavenge several RONS
that can cause lipid peroxidation (e.g., hydroxyl radical,
lipid peroxyl radical, peroxynitrite, and hydrogen perox-
ide) directly and indirectly through enzymatic reactions
(54). In addition, GSH is a substrate for glutathione
peroxidase, which catalyzes the reduction of peroxides,
such as hydrogen peroxide and lipid hydroperoxides (54).
Another potential mechanism through which spirulina
decreased lipid peroxidation might be the increased
content of F-linolenic acid in spirulina (33). Indeed, it
has been found that an increased ratio of F-linolenic acid
to arachidonic acid is capable of attenuating the biosyn-
thesis of arachidonic acid metabolites (i.e., prostaglandins,
leukotrienes, and platelet-activating factor) and exerts an
anti-inflammatory effect (9,19). Decreased inflammation
via this route might have decreased the production of
superoxide, hydrogen peroxide, and hypochlorous acid
by the activated neutrophils (10) leading to less lipid per-
oxidation after spirulina supplementation.
Regarding the remaining indices of redox status (protein
carbonyls, catalase, and TAC), all increased immediately
and 1 h after exercise indicating oxidative stress. All redox
status indices returned to their preexercise values at 24 h.
Studies that have investigated the effects of aerobic exercise
on serum protein carbonyls generally have reported
increases similar to ours lasting up to 6 h of recovery (8,29).
Evidence addressing the efficacy of antioxidant supple-
mentation to decrease oxidative stress remains ambiguous.
For example, it has been shown that supplementation for
4 wk with vitamin E prevented the increase of lipid
peroxidation after exercise (46). In addition, supplementa-
tion for 2 wk with vitamins C and E attenuated the rise in
protein oxidation after exercise (8). On the contrary,
supplementation for 6 wk with vitamin C, vitamin E, and
A
-carotene did not prevent the exercise-induced increase of
lipid peroxidation (20). Moreover, supplementation for
5 wk with artichoke extract did not attenuate oxidative
damage to erythrocytes after exercise (45). These differences
in results may be related, in part, to the different concentra-
tion of the antioxidants and the combination of ingredients.
Mobilization of tissue antioxidant stores into plasma,
such as uric acid (13), is probably one mechanism re-
sponsible for the marked increase (and not decrease, as
might be expected intuitively) of TAC after exercise. This is
a widely accepted phenomenon that helps maintain or even
increase serum antioxidant status in times of need (39).
Increased catalase activity after exercise also could have
contributed to the increased TAC. Nevertheless, this
increase in the antioxidant capacity of serum did not prove
efficient at inhibiting the increase in lipid and protein
oxidation in the blood. Most studies agree that exercise
increases TAC for some hours after exercise (3,53). Perhaps
the increased TAC could mean that the plasma gets
enriched with antioxidant molecules that need to be trans-
ported into tissues where they can provide protection.
CONCLUSIONS
Many positive claiMSS for spirulina are based on
research done on individual nutrients that spirulina contains,
such as various antioxidants, rather than on direct research
using spirulina. This is one of the few studies where
humans were supplemented with spirulina. We report for
the first time that supplementation of spirulina for 4 wk
increased exercise performance, possibly through an in-
crease in fat oxidation rate, and increased GSH levels. The
reasons behind the enhanced performance and increased fat
oxidation after spirulina supplementation are poorly under-
stood, and more research is needed to elucidate this.
Particularly, the effect of spirulina on mitochondrial
function and Aoxidation in conjunction with inflammation
and oxidative stress requires further investigation.
This study was supported by funds from the Center of Research
and Technology – Thessaly.
The results of the present study do not constitute endorsement
by American College of Sports Medicine.
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