Original Paper.

Physical Education and Sport, 52, 96 - 99, 2008

DOI: 10.2478/v10030-008-0022-6

Two doses of caffeine do not increase the risk of

Authors’ contributions: exercise-induced muscle damage or leukocytosis
A Study design
B Data collection
C Statistical analysis
Natália S. Vimercatti B E G, Paulo V. C. Zovico B G, Andréa S. Carvalho B G, Juliano
D Data interpretation G. Barreto B E D, Marco Machado A–F
E Literature search
F Manuscript preparation Laboratory of Physiology and Biokinetics (UNIG Campus V), Itaperuna RJ, Brazil
G Funds collection

Summary

Study aim: To examine the effects of caffeine supplementation on leukocyte count and
muscle damage markers in responses to moderate exercise.
Material and methods: A group of 15 male subjects participated in a placebo controlled,
double-blind, cross-over experiment consisting of placebo or caffeine (4.5 or 5.5 mg/kg
body mass) ingestion and performing a 60-min treadmill exercise at 65% ‡O2max. Blood
samples were collected before and immediately after exercise for leukocyte counts, and
creatine kinase (CK), lactate dehydrogenase (LDH), aspartate (AST) and alanine (ALT)
aminotransferase activities. ANOVA and post-hoc Tukey’s test were used in data proc-
essing.
Results: Leukocyte count, CK, LDH and, to some extent, AST activities increased in re-
sponse to exercise but all the studied variables showed no caffeine-induced increases.
Conclusion: Caffeine supplementation in the studied range had no effect on immune re-
sponses or muscle cell integrity.

Key words Caffeine – Creatine kinase – Exercise – Muscle damage

Introduction age [2,4,15]. Tidball [18] reported recently that muscle

Caffeine (1,3,7-trimethyl-xanthine) is the most widely
used stimulant. It was removed from the list of prohib-
ited substances by the World Anti-Doping Agency in
2004 and has been increasingly used as an ergogenic
supplement by athletes and recreational practitioners.
This occurred even though there is general consensus
from research findings that caffeine improves the per-
formance of prolonged, continuous exercise [5,7]. Sup-
plementation with caffeine is also known to decrease
muscular pain perception, effort perception, and neuro-
muscular reaction time which can further enhance the
performance [7,10,17].
Exercise-induced micro-damage is described as mus-
cle membrane disruption followed by an inflammatory
process. This brings about increases in serum activities
of enzymes, e.g. creatine kinase (CK), lactate dehydro-
genase (LDH), alanine (ALT) or aspartate (AST) ami-
notransferase, which serve as markers of muscle dam-
micro-damage is an inflammatory factor during and
after the exercise.
According to Bassini-Cameron et al. [1], the effects
of caffeine on leukocytes may intensify the exercise-
induced muscle damage. However, some authors do not
share that opinion [20]. A pre-exercise caffeine inges-
tion leads to an enhanced activation of both the hypo-
thalamic-pituitary-adrenal axis and the autonomic nerv-
ous system [7,8] which, in turn, may affect the immune
responses to exercise [19].
Caffeine and caffeine-based substances have been
increasingly used as ergogenic supplements by recrea-
tional and professional physical activity practitioners,
but the effects of those substances on human immune
system during physical activity and on the exercise-
induced micro-damage are still obscure. Therefore, the
aim of this study was to examine the effects of caffeine
supplementation on leukocyte count and muscle damage
markers in response to a moderate-intensity exercise.

Dr Marco Machado, Laboratório de Fisiologia e Biocinética (UNIG – Campus V), Curso de Educação
Author’s address Física, Universidade Iguaçu (UNIG), BR 356 - Km 02 Itaperuna, RJ, Brazil CEP 28.300-000
marcomachado1@gmail.com
Caffeine, muscle damage, leukocytosis
Material and Methods
A group of 15 male subjects aged 19 ± 1 years vol-
unteered to participate in the study. They were healthy,
physically active (trained up to 3 times weekly), used no
therapeutic agents, dietary supplements or anabolic ster-
oids. All subjects submitted their written consents to par-
ticipate and the study was approved by the local com-
mittee of ethics.
A double-blind, placebo-controlled cross-over design
was used. Each subject performed 3 experimental trials,
each 2 weeks apart. The subjects abstained from ingest-
ing caffeine, xanthines or other substances that could
mask the results for 12 h preceding blood collections.
The subjects reported at the laboratory in the morn-
ing (8:00 – 9:00) after an overnight fast (8 – 12 h). The
start time allocated to each subject remained the same in
all trials in order to avoid circadian variance. After the
morning blood withdrawal, the subjects consumed a stan-
dard breakfast (about 50 g of bread, 20 g of cheese and
200 ml of skimmed milk), then caffeine (or placebo) was
administered and 35 min later they were subjected to a
10-min warm up (jogging, joint mobilisation and stretch-
ing). The warm-up was followed by a 60-min treadmill
exercise at the individually pre-determined workload
equal to 65% ‡O2max.
Caffeine (Jilin Shulan, China) or cellulose (Gujarat
Microwax, India) were administered by random alloca-
tion. Caffeine dosage was equal to 4.5 or 5.5 mg/kg body
mass, the doses being within the range 3 – 9 mg/kg ad-
ministered 30 – 60 min pre-exercise reported to improve
performance [7]. In the control trial, the subjects received
capsules containing 500 mg cellulose, indistinguishable
from those containing caffeine.
Blood was sampled from the antecubital vein in the
morning and immediately after the exercise into two tubes:
one containing EDTA for haematological examination,
the other was centrifuged and serum collected. Serum
samples were immediately frozen and stored at -70°C.
The following haematological variables were recorded
using an automated analyser (Cobas Mira S Plus, Roche):
hematocrit, erythrocyte counts (RBC), haemoglobin (Hb)
and leukocyte counts (WBC). The activities of creatine
kinase (CK), lactate dehydrogenase (LDH), alanine amino-
transferase (ALT) and aspartate aminotransferase (AST)
were assayed using specific commercial kits (Biotécnica,
Brazil).
Three-way ANOVA (supplement, exercise, subjects)
with Bonferroni adjustment was used in data analysis,
the level of p≤0.05 being considered significant.
97
Results
Mean values (±SD) of age, somatic and haemato-
logical variables are shown in Table 1. All haematologi-
cal indices (including MCV, MCH and MCHC, not re-
ported here) were within normal limits and were fairly
stable throughout the experiment. The aerobic power of
subjects expressed by the ‡O2max was fairly high.
Table 1. Mean values (±SD) of somatic and haematolo-
gical variables recorded in young male subjects (n = 15)
Reference
Variable Mean ± SD
ranges [9]
Age (years) 19.1 ± 1.0 –
Body height (cm) 173 ± 6 –
Body mass (kg) 64.7 ± 8.8 –
‡O2max (ml/kg/min) 52.0 ± 2.7 –
Hematocrit (%) 44.2 ± 2.7 37.0 - 49.0
6 3
Erythrocyte (×10 /mm ) 4.9 ± 0.4 4.5 - 5.3
Haemoglobin (%) 15.9 ± 0.9 13.0 - 18.0
Leukocyte count per mm³ 6433 ± 1221 5000 – 9000
The changes in leukocyte counts and enzyme activi-
ties in all trials are presented in Fig. 1 (next page). The
post-exercise leukocyte counts were significantly (p<0.05)
higher than the pre-exercise ones in all trials, caffeine
being ineffective in that respect. Significant post-exercise
increases were also observed in case of creatine kinase,
lactate dehydrogenase and aspartate aminotransferase.
Again, caffeine administration did not significantly in-
crease any of the studied variables.
Discussion
Previous studies suggest that caffeine ingestion has a
hypoalgesic effect on muscle during high-intensity ex-
ercise [10,12] and enhances the risk of muscle damage
in soccer players [1]. This prompted us to investigate
into the effects of two doses of caffeine supplementation
on leukocyte count and 4 markers of muscle damage
before and after a moderate exercise.
Many authors reported exercise-induced leukocyto-
sis [13,16] the results being consistent with our findings.
The immune response to exercise includes alterations
in the circulating white blood cells counts and differen-
tial leukocyte subset trafficking. Interaction between
cytokines, catecholamine, cortisol, prostaglandins and
98 N. S. Vimercatti et al.

leukotrienes released following muscle damage and the immune cells to migrate to the circulatory system [14,
products of cellular metabolism stimulates bone marrow 18,21].
U/L U/L U/L
9000 400 550
* * WBC CK LDH
8500 * 380
500
8000 360 * * * *
7500 340 * 450
7000 320 400
Pre Pre Pre
6500 300
6000
Post
280
* Post 350 Post

5500 260 300

5000 240
250
4500 220
4000 200 200
0 4.5 5.5 0 4.5 5.5 0 4.5 5.5
Caffeine dose (mg / kg) Caffeine dose (mg / kg) Caffeine dose (mg / kg)
U/L U/L
32 22 ALT
AST
30 20
*
28 18
Pre Pre
26 16
* Post Post
24 14

22 12

20 10
0 4.5 5.5 0 4.5 5.5
Caffeine dose (mg / kg) Caffeine dose (mg / kg)

Fig. 1. White blood cell count and activities of enzymes in serum of young athletes following oral administration of
caffeine
Legend: WBC – Leukocyte count per mm3; CK – Creatine kinase activity; LDH – Lactate dehydrogenase activity; AST – Aspar-
tate aminotransferase; ALT – Alanine aminotransferase; * Significant (p<0.05) difference between the pre- and post-exercise value

The levels of markers recorded in this study were in-
dicative of muscle damage following exercise of mod-
erate intensity. The activities of CK and LDH were con-
sistent with other reports; Pettersson et al. [15] reported
increases in CK and LDH activities following resistance
exercises, the increases being highest 24 – 72 h post-
exercise. The baseline CK activity in the present study
was higher than that proposed for the general population
[9], but the subjects were physically active, engaging in
light-to-moderate physical activity. It would thus seem
recommendable to revise the reference intervals for that
enzyme. For example, Mougious [11] suggested that the
cut-off values for physically active males and females
be 491 and 252 U/L, respectively.
Bassini-Cameron et al. [1] demonstrated a synergis-
tic effect of caffeine and exercise; following a simulated
soccer match, the subjects performed a very intense ex-
ercise (Yo-yo test). Since caffeine could have played a
role in delaying fatigue and exerted a hypoalgesic effect
on the muscle, this hypothesis should have been reflected
by a longer time of work until exhaustion in the caffeine
group but these data were not reported in that study.
The ALT and AST aminotransferases are among the
most reliable markers of hepatocellular injury or necro-
sis; also physical exertion is known to bring about tran-
sient elevations in their activities [3,4]. In fact, the total
content of AST and ALT in serum represents the pas-
sage of muscle and hepatic enzymes into circulation;
Pettersson et al. [15] warned about imposing relevant
restrictions on exercise practice prior to and during drug
clinical studies. Our results concerning the activities of
aminotransferases are consistent with the reports of
other authors that AST and ALT activities increased
only 24 – 72 h following an injury [15].
No significant exercise-related differences in either
hematocrit, erythrocyte counts or haemoglobin were
found in this study which was indicative of a lack of
changes in blood volume. This finding is important be-
cause haemoconcentration or haemodilution could have
resulted in erroneous data interpretation [6]. In our opin-
ion, the increases in leukocyte count, and in CK and LDH
activities, were induced by muscle stress and injury and
not haemoconcentration.
Caffeine, muscle damage, leukocytosis
Summing up, the study indicated that moderate ex-
ercise induced an increase in leukocyte population and
increases in serum markers of muscle damage. On the
other hand, caffeine supplementation at dosages of 4.5
or 5.5 g/kg had no significant effect in a highly reliable
cross-over design which may suggest no harmful effects
of caffeine in that respect.
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Received 24.09.2008
Accepted 24.11.2008
© University of Physical Education, Warsaw, Poland
Acknowledgements
Thanks are due to Pierre Augusto Victor da Silva (UNIG
Pharmacy-School) for preparation of supplements and to
Wilkes de Oliveira for assistance in blood analyses