Original Paper.

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

DOI: 10.2478/v10030-008-0022-6

Authors contributions: A Study design B Data collection C Statistical analysis D Data interpretation E Literature search F Manuscript preparation G Funds collection

Two doses of caffeine do not increase the risk of exercise-induced muscle damage or leukocytosis
Natlia S. Vimercatti B E G, Paulo V. C. Zovico B G, Andra S. Carvalho B G, Juliano G. Barreto B E D, Marco Machado AF
Laboratory of Physiology and Biokinetics (UNIG Campus V), Itaperuna RJ, Brazil

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 Tukeys test were used in data processing. Results: Leukocyte count, CK, LDH and, to some extent, AST activities increased in response to exercise but all the studied variables showed no caffeine-induced increases. Conclusion: Caffeine supplementation in the studied range had no effect on immune responses or muscle cell integrity.

Key words

Caffeine Creatine kinase Exercise Muscle damage

Introduction
Caffeine (1,3,7-trimethyl-xanthine) is the most widely used stimulant. It was removed from the list of prohibited 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 performance of prolonged, continuous exercise [5,7]. Supplementation with caffeine is also known to decrease muscular pain perception, effort perception, and neuromuscular reaction time which can further enhance the performance [7,10,17]. Exercise-induced micro-damage is described as muscle membrane disruption followed by an inflammatory process. This brings about increases in serum activities of enzymes, e.g. creatine kinase (CK), lactate dehydrogenase (LDH), alanine (ALT) or aspartate (AST) aminotransferase, which serve as markers of muscle dam-

age [2,4,15]. Tidball [18] reported recently that muscle 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 exerciseinduced muscle damage. However, some authors do not share that opinion [20]. A pre-exercise caffeine ingestion leads to an enhanced activation of both the hypothalamic-pituitary-adrenal axis and the autonomic nervous 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 recreational and professional physical activity practitioners, but the effects of those substances on human immune system during physical activity and on the exerciseinduced 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, Laboratrio de Fisiologia e Biocintica (UNIG Campus V), Curso de Educao Authors address Fsica, Universidade Iguau (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 volunteered to participate in the study. They were healthy, physically active (trained up to 3 times weekly), used no therapeutic agents, dietary supplements or anabolic steroids. All subjects submitted their written consents to participate and the study was approved by the local committee 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 ingesting caffeine, xanthines or other substances that could mask the results for 12 h preceding blood collections. The subjects reported at the laboratory in the morning (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 standard 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 stretching). 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 allocation. Caffeine dosage was equal to 4.5 or 5.5 mg/kg body mass, the doses being within the range 3 9 mg/kg administered 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 -70C. 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 aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed using specific commercial kits (Biotcnica, Brazil). Three-way ANOVA (supplement, exercise, subjects) with Bonferroni adjustment was used in data analysis, the level of p0.05 being considered significant.

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Results
Mean values (SD) of age, somatic and haematological variables are shown in Table 1. All haematological indices (including MCV, MCH and MCHC, not reported 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 haematological variables recorded in young male subjects (n = 15) Variable Mean SD Reference ranges [9] 37.0 - 49.0 4.5 - 5.3 13.0 - 18.0 5000 9000

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 6 3 Erythrocyte (10 /mm ) 4.9 0.4 Haemoglobin (%) 15.9 0.9 Leukocyte count per mm 6433 1221

The changes in leukocyte counts and enzyme activities 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 increase any of the studied variables.

Discussion
Previous studies suggest that caffeine ingestion has a hypoalgesic effect on muscle during high-intensity exercise [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 leukocytosis [13,16] the results being consistent with our findings. The immune response to exercise includes alterations in the circulating white blood cells counts and differential leukocyte subset trafficking. Interaction between cytokines, catecholamine, cortisol, prostaglandins and

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leukotrienes released following muscle damage and the products of cellular metabolism stimulates bone marrow
9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 0 4.5 5.5 Caffeine dose (mg / kg)
32 30 28 26 24 22 20 0 4.5 5.5 Caffeine dose (mg / kg) Pre U/L

N. S. Vimercatti et al.
immune cells to migrate to the circulatory system [14, 18,21].
CK
550 500 U/L

U/L

WBC

400 380 360 340 320 300 280 260 240 220 200

U/L

LDH

*
Pre Post

*
Pre

450 400 350 300 250 200

Pre Post

Post

4.5 5.5 Caffeine dose (mg / kg)

4.5 5.5 Caffeine dose (mg / kg)

AST

22 20 18

U/L

ALT

Post

16 14 12 10 0 4.5 5.5 Caffeine dose (mg / kg)

Pre Post

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 Aspartate 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 indicative of muscle damage following exercise of moderate intensity. The activities of CK and LDH were consistent with other reports; Pettersson et al. [15] reported increases in CK and LDH activities following resistance exercises, the increases being highest 24 72 h postexercise. 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 synergistic effect of caffeine and exercise; following a simulated soccer match, the subjects performed a very intense exercise (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 necrosis; also physical exertion is known to bring about transient elevations in their activities [3,4]. In fact, the total content of AST and ALT in serum represents the passage 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 because haemoconcentration or haemodilution could have resulted in erroneous data interpretation [6]. In our opinion, 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

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12. O'Connor P.J., R.W.Motl, S.P.Broglio, M.R.Ely (2004) Dose-dependent effect of caffeine on reducing leg muscle pain during cycling exercise is unrelated to systolic blood pressure. Pain 109:291-298. 13. Peake J.M., K.Suzuki, G.Wilson, M.Hordern, K.Nosaka, L.Mackinnon, J.S.Coombes (2005) Exercise-induced muscle damage, plasma cytokines, and markers of neutrophil activation. Med.Sci.Sports Exerc. 37:737-745. 14. Pedersen B.K., A.D.Toft (2000) Effects of exercise on lymphocytes and cytokines Br.J.Sports Med. 34:246-251. 15. Pettersson J., U.Hindorf, P.Persson, T.Bengtsson, U.Malmqvist, V.Werkstrm, M.Ekelund (2007) Muscular exercise can cause highly pathological liver function tests in healthy men. Br.J.Clin.Pharmacol. 65:253-259. 16. Scharhag J., T.Meyer, H.H.W.Gabriel, B.Schlick, O.Faude, W.Kindermann (2005) Does prolonged cycling of moderate intensity affect immune cell function? Br.J.Sports Med. 39: 171-177. 17. Schneiker K.T., D.Bishop, B.Dawson, L.P.Hacket (2006) Effects of caffeine on prolonged intermittent-sprint ability in team-sport athletes. Med.Sci.Sports Exerc. 38:578-585. 18. Tidball J.G. (2008) Inflammation in skeletal muscle regeneration. In: S.Schiaffino, T. Partridge (eds.), Skeletal Muscle Repair and Regeneration, Springer Science+Business Media. 19. Tsigos C., G.P.Chrousos (2002) Hypothalamicpituitary adrenal axis, neuroendocrine factors and stress. J.Psychosom. Res. 53:865-871. 20. Walker G.J., O.Finlay, H.Griffiths, J.Sylvester, M.Williams, N.C.Bishop (2007) Immunoendocrine response to cycling following ingestion of caffeine and carbohydrate. Med. Sci.Sports Exerc. 39:1554-1560. 21. Zaldivar F., J.Wang-Rodriguez, D.Nemet, C.Schwindt, P.Galassetti, P.J.Mills, L.D.Wilson, D.M.Cooper (2006) Constitutive pro- and anti-inflammatory cytokine and growth factor response to exercise in leukocytes. J.Appl.Physiol. 100: 1124-1133. 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

Summing up, the study indicated that moderate exercise 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.

References
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