1010

Immunology

Lymphocyte and Cytokines after Short Periods of Exercise

Authors

J. Prestes 1, C. K. de O. Ferreira 1, R. Dias 1, A. B. Frollini 1, F. F. Donatto 1, M. F. Cury-Boaventura 2, M. G. Guereschi 1, T. C. Pithon-Curi 2, R. Verlengia 1, A. C. Palanch 1, R. Curi 2, C. R. Cavaglieri 1
1 2

Affiliations

Health Science Faculty, Methodist University of Piracicaba, Piracicaba, Brazil Department of Physiology and Biophysics, University of So Paulo, So Paulo, Brazil

The purpose of this study was to verify the effects of short periods of exercise of different intensity on lymphocyte function and cytokines. Thirty Wistar rats, 2 months old, were used. They were divided into five groups of six rats: a sedentary control group; a group exercised for 5 minutes at low intensity (5 L); a group exercised for 15 minutes at low intensity (15 L); and groups exercised at moderate intensity (additional load of 5% of body weight) for 5 minutes (5 M) or for 15 minutes (15 M). The parameters measured were: total leukocytes, neutrophils, lymphocytes, monocytes, lymphocytes from lymph nodes, serum cytokines (IL-2, IL-6 and TNF-a), lymphocyte mito-

Introduction
!

accepted after revision May 5, 2008 Bibliography DOI 10.1055/s-2008-1038737 Published online July 3, 2008 Int J Sports Med 2008; 29: 1010 1014 Georg Thieme Verlag KG Stuttgart New York ISSN 0172-4622 Correspondence Jonato Prestes Methodist University of Piracicaba Health Sciences Faculty Major Jos Incio st, n. 2400 13560161 So Carlos Brazil Phone: + 55 (16) 33 76 13 90 Fax: + 55 (19) 31 24 15 15 jonatop@gmail.com

The present day lack of time, excessive work journeys and stress have led many individuals to perform short bouts of exercise (5 25 minutes). Exercise intensity, duration and frequency, and individual levels of physical fitness are important factors in determining immune response to physical effort. Exercise may stimulate or lower immune function [18]. Regular physical activity performed at a moderate intensity is associated with a decreased sensitivity to upper respiratory tract infections (URTIs) [18]. However, high-intensity training, performed typically by athletes, may increase sensitivity to URTIs [19]. Temporary immunosuppression, termed the open window period, when athletes are more susceptible to infection occurs after exhaustive bouts of exercise [23]. Intensive training may reduce lymphocyte function or accelerate the process of apoptosis in these cells [23]. Strenuous and lengthy exercise induces leukocytosis in direct proportion to exercise intensity, and this rise in cell count occurs mainly in neutrophils [1, 9]. The number of

monocytes and lymphocytes are also increased, but by a smaller amount [9], and although the number of lymphocyte subpopulations increases, the level of natural killer cells (NK) demonstrates the greatest alteration [23]. This transitory lymphocytosis is attributed to exercise-induced epinephrine release [23]. Exercise induces alterations in tumor necrosis factor-a (TNF-a) [15], interleukin-6 (IL-6) [21], interferons and other cytokines [16]. IL-6 is a key proinflammatory cytokine in the acute phase of inflammatory response [21]. The demands of exercise and the resulting reduction in the activity of NK cells during the recovery period are related to decreases in the expression of IL-2 membrane receptors in NK cells [14]. In adults, increases in circulating TNF-a (proinflammatory cytokine) induced by exhaustive exercise have been demonstrated [21]. Programmed cell death, apoptosis, is an important mechanism for regulating the immune response [20]. Exhaustive exercise may induce increases in leukocyte apoptosis [26]. Recently, Tuan et al. [27] observed that 3 consecutive days

Prestes J et al. Lymphocyte and Cytokines Int J Sports Med 2008; 29: 1010 1014

Downloaded by: Dot. Lib Information. Copyrighted material.

Key words " physical exercise l " leukocytes l " immune function l " lymphocytes l " cytokines l

Abstract
!

chondrial transmembrane potential, viability and DNA fragmentation. ANOVA two way followed by Tukeys post hoc test (p 0.05) was used. The exercised groups exhibited a significant increase in total leukocytes, tissue and circulating lymphocytes in comparison with the control group. There was a significant decrease in lymphocyte viability and decrease in DNA fragmentation for the 15 M group when compared with the control. There was a decrease in the level TNF-a in the 5 M and 15 M groups. Short-term, low- and moderate-intensity exercise may be considered for sedentary individuals beginning to exercise, since no deleterious alterations were observed in lymphocyte function.

Immunology

1011

O2max for 30 min of short-term, high-intensity exercise (85 % of V every day) induced increase in monocytes, lymphocytes and neutrophils apoptosis in trained individuals. Concomitantly, the authors also showed an increase in TNF-a plasma levels and its soluble Fas ligand apoptosis receptor (sFasL). In a study of our group, with 5 and 15 minutes of acute low- and moderate-intensity exercise, no neutrophil apoptosis was verified in sedentary rats. Interestingly, after 5 and 15 minutes of moderate-intensity exercise, TNF-a levels were reduced [3]. Based on the abovementioned studies, it was resolved to investigate the effects of short-term, low- and moderate-intensity exercise on leukocyte counts, lymphocyte apoptosis, their relationship with cytokines levels and immune responses.

Serum cytokine determination

Measurements of IL-2, IL-6 and TNF-a in plasma were made by ELISA using the R & D Systems Quantikine High Sensitivity kit (R & D Systems, Minneapolis, MN, USA) for each cytokine. The intra-assay coefficient of variance (CV) was 4.1 10%, the inter-assay CV was 6.6 8%, and the sensitivity was 0.0083 pg/mL.

Circulating lymphocyte mitochondrial transmembrane potential

Cells were centrifuged at 4 8C for 15 min at 1000g, and the pellet was resuspended in 1000 L of PBS. Rhodamine 123 (5 M) was added and cells were incubated for 15 min at 37 8C in the dark. Cells were washed twice with PBS and incubated for 30 min at 30 8C in the dark. Fluorescence of 10 000 events was determined by flow cytometry using the FL1 channel (green fluorescence 530/30 nm).

Materials and Methods

!

Animals
Two-month-old male Wistar rats (Rattus novergicus var. albinus, Rodentia, Mammalia) with a mean weight of 200 g were used. The animals had free access to water and chow. The animals were kept in collective cages (3 rats per cage) at a constant temperature of 23 ( 2) 8C, and a cycle of 12 hours light/12 hours dark, with light from 06 : 00 h to 18 : 00 h. The research was approved by the Federal University of So Carlos Committee of Experimental Animals. The animals were divided into five groups (six animals per group): a sedentary control group, groups exercised at low intensity for 5 minutes (5 L), or 15 minutes (15 L), and groups exercised at moderate intensity for 5 minutes (5 M), or 15 minutes (15 M). The exercised groups performed a single acute exercise session.

Circulating lymphocyte viability

Lymphocytes were centrifuged at 1000 g for 15 min at 4 8C, and the pellet obtained was resuspended in 500 L of PBS, 50 L of propidium iodide solution (50 mg/mL in PBS) was added, and cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). In cells that have lost membrane integrity, propidium iodide binds to DNA by intercalating between bases, with little or no sequence preference. Fluorescence was measured using the FL2 channel (orange-red fluorescence, 585/42 nm). Cells with propidium iodide fluorescence were evaluated by CellQuest software (Becton Dickinson).

Circulating lymphocyte DNA fragmentation

DNA fragmentation was analyzed by flow cytometry after staining DNA with propidium iodide. Cells were resuspended in a solution of detergents that permeabilize cells, and the dye was promptly incorporated into DNA. Cells were centrifuged at 1000 g for 15 min at 4 8C. The pellet was resuspended gently in 300 L of hypotonic solution that contained 50 g/mL of propidium iodide, 0.1% sodium citrate, and 0.1% Triton X-100. Cells were then incubated for 2 h at 4 8C.

Physical exercise
The exercise was swimming, accomplished between 14 : 00 17: 00 h in a tank maintained at 30 ( 2) 8C. The animals were sacrificed immediately after exercise. The low-intensity groups did not carry an additional load. At moderate intensity, the animals carried a load of 5% of body weight on their backs, which corresponds to intensity below the point of inflection of the lactate threshold curve. In swimming exercise, Wistar rats achieve the lactate threshold at a load of 5.2 % of their body weight. In the present study, the exercise with the load of 5 % was next to the lactate threshold, but below it, characterizing moderate intensity [28].

Statistical analysis
Data are presented as mean SEM (standard error of the mean). Differences between the control and exercised groups were analyzed by two-way analysis of variance (ANOVA) (considering the intervening variables intensity duration). When a significant value was observed, Tukeys post hoc test was used with a significance level of p 0.05.

Total circulating levels of leukocytes, neutrophils, lymphocytes and monocytes

The count of total leukocytes was accomplished with Turkey liquid and counting was performed in a Neubauer chamber. The circulating levels of neutrophils, lymphocytes and monocytes were measured under an optical microscope using a blood smear stained with Giemsa-May-Grnwald.

Results
!

Tissue lymphocytes
All mesenteric lymph nodes were removed, placed between two cylinders and compressed (maceration) with the use of circular movements in order to obtain the isolated and integral lymphocytes. The numbers of lymphocytes were determined by the application of Triplan blue; the cell count was performed in the Neubauer counting chamber under an optical microscope.

A significant increase in total leukocytes was observed for the groups exercised at low and moderate intensities for 5 minutes (5 L and 5 M) or 15 minutes (15 L and 15 M) when compared with the control. A significant increase in leukocytes was observed for the 5 M group when compared with the 5 L group. A significant increase in circulating neutrophils, lymphocytes and monocytes was detected for the exercised groups in comparison with the control. When exercised groups were compared, the 15 M group showed a higher level of neutrophils when compared with the 15 L group. 5 M showed a higher lymphocyte level than that of group 5 L. The level of circulating monocytes increased signifi" Table 1). cantly more in the 15 L compared to 15 M group (l

Prestes J et al. Lymphocyte and Cytokines Int J Sports Med 2008; 29: 1010 1014

Downloaded by: Dot. Lib Information. Copyrighted material.

1012

Immunology

Table 1 Total leukocytes and circulating neutrophils, lymphocytes and monocytes levels Total cells count 106
Total leukocytes Neutrophils Lymphocytes Monocytes

C
4.12 0.17 1.31 0.0012 2.65 0.0008 0.14 0.0004

5L
8.56 1.06* 2.86 0.0161* 5.24 0.0166* 0.46 0.0025*

15 L
9.5 0.91* 1.8 0.01* 7.03 0.008* 0.66 0.004*

5M
12.64 0.9*# 2.78 0.007* 9.33 0.0072*# 0.53 0.0018*

15 M
11.6 0.6* 3.59 0.0053*# 7.7 0.0061* 0.3 0.0024*#

Values are mean SEM. C, control group; 5 L, 5 minutes low-intensity exercise; 15 L, 15 minutes low-intensity exercise; 5 M, 5 minutes moderate-intensity exercise; 15 M, 15 minutes moderate-intensity exercise. *Significant difference as compared to the control group. #Significant difference between exercised groups at the same duration (p 0.05)

Table 2 IL-2, IL-6 and TNF-a plasma levels Cytokines (pg/mL)

IL-2 IL-6 TNF-a

C
56.2 7.01 267.88 9.86 5.46 0.20

5L
53.14 2.54 262.54 17.67 4.84 0.27

15 L
48.97 2.68 279.88 13.01 5.25 0.20

5M
48.4 2.88 283.21 28.62 4.18 0.16*

15 M
49.46 1.63 294.54 12.66 4.2 0.08*#

Values are mean SEM. C, control group; 5 L, 5 minutes low-intensity exercise; 15 L, 15 minutes low-intensity exercise; 5 M, 5 minutes moderate-intensity exercise; 15 M, 15 minutes moderate-intensity exercise. *Significant difference as compared to the control group. #Significant difference between exercised groups at the same duration (p 0.05)

Groups 5 L, 5 M and 15 M exhibited significant increases in the number of tissue lymphocytes in comparison with the control group. A higher number of tissue lymphocytes were observed " Fig. 1 A). for 15 M when compared with 15 L (l TNF-a showed a significant decrease in 5 M and 15 M groups as compared to control. Between exercised groups, there was a significant decrease in TNF-a for 15 M in comparison with 15 L. No change was found in the level of IL-2 or IL-6 in the exercised " Table 2). groups (l A significant decrease in lymphocyte viability was detected for " Fig. 1 B). There was no 15 M in comparison with the control (l significant alteration in the lymphocytes mitochondrial trans" Fig. 2 A). A decrease in lymphocytes membrane potential (l DNA fragmentation was observed for the 15 L, 5 M and 15 M groups when compared with the control. A decrease in lymphocytes DNA fragmentation was observed for 15 M group in com" Fig. 2 B). parison with 15 L (l

Discussion
!

Many studies have been conducted to analyze the effects of lengthy exercise (marathon) on the immune response. However, the effects of short periods of exercise are not very clear. This is the first study to analyze the effects of 5 or 15 minutes exercise at low and moderate intensity, on immune function. The findings of the present research provide primary insights into the understanding of immune system modifications after acute brief exercise. Leukocytosis occurs in response to acute brief exercise [5]. The effects of exercise on the increase of circulating leukocytes are mediated, in part, by activation of the sympathetic nervous system [5] and consequently, acute increase in the levels of catecholamines, especially during short-duration exercise [13]. Similarly, in the present study, leukocytosis was observed in exercised groups when compared with the control. The moderate intensity of exercise induced greater elevation of the number of circulating leukocytes. This intensity dependence has been observed in other studies with short periods of exercise [1, 9].

Fig. 1 A and B Numbers of lymphocytes from the mesenteric lymph nodes (A) and circulating lymphocytes viability (B). The results are the average SEM. *Significant difference as compared to the control group; # significant difference compared between the same duration groups; p 0.05. C, control group; 5 L, 5 minutes low-intensity exercise; 15 L, 15 minutes low-intensity exercise; 5 M, 5 minutes moderate-intensity exercise; 15 M, 15 minutes moderate-intensity exercise.

Lymphocytosis was detected in groups exercised at low and moderate intensity. The levels of lymphocytes showed a depen-

Prestes J et al. Lymphocyte and Cytokines Int J Sports Med 2008; 29: 1010 1014

Downloaded by: Dot. Lib Information. Copyrighted material.

Immunology

1013

Fig. 2 A and B Circulating lymphocyte mitochondrial transmembrane potential (MTP) (A) and DNA fragmentation (B). The results are the average SEM. *Significant difference as compared to the control group; # significant difference compared between the same duration groups, p 0.05. C, control group; 5 L, 5 minutes low-intensity exercise; 15 L, 15 minutes low-intensity exercise; 5 M, 5 minutes moderate-intensity exercise; 15 M, 15 minutes moderate-intensity exercise.

dence on exercise intensity, with higher levels of lymphocytes in the 5 M group when compared with the 5 L group. Almost 2/3 of total lymphocytes released to the circulation during exercise come from the spleen [17]. The secondary lymphoid organs may be responsible for the increase of circulating cells [4]. In the present study, the higher circulating lymphocytes counts for the exercised groups were matched with an increase in tissue lymphocytes. Apoptosis and necrosis were detected in lymphocytes after acute intense aerobic exercise [2]. A change in mitochondrial transmembrane potential, which provides the conducting force for ATP formation, represents an obligatory step in the induction of apoptotic death [11]. After exhaustive exercise, mitochondrial membrane depolarization occurs in rat neutrophils [12] and in mouse intestinal lymphocytes [25]. Thymocyte apoptosis was evidenced by DNA fragmentation in rats that performed two treadmill runs to exhaustion [2]. It has been shown that high-intensity treadmill running was associated with expression of pro-apoptotic proteins and reduction in the expression of the antiapoptotic protein in rats intestinal lymphocytes [26].

Thymocyte apoptosis was not observed in mice submitted to submaximum treadmill exercise during 90 minutes [8]. Moderate exercise did not induce circulating lymphocyte apoptosis, as shown in the present study. With low-intensity exercise for 15 minutes, moderate-intensity for 5 and 15 minutes, a decrease in the lymphocytes with DNA fragmentation was observed, indicating that, despite the decrease in viability, fewer lymphocytes were in apoptosis as a result of DNA fragmentation. It is difficult to draw any final conclusion from these results, and they should be treated with caution. Few studies on lymphocyte apoptosis following brief periods of exercises have been reported. No significant difference in mitochondrial transmembrane potential was found for the groups. These results differ from other studies [12, 25], which used a prolonged and different type of exercise from that in this study. If fewer lymphocytes were in apoptosis by DNA fragmentation, it would be expected that the mitochondrial transmembrane potential would not change. Mitochondrial depolarization precedes nuclear signals of apoptosis activation of endogenous endonucleases and caspases, which result in irreversible fragmentation of cell DNA [11]. Furthermore, short-term, high-intensity exercise can lead to a significant and prolonged dysfunction of the mitochondrial energy status of peripheral blood leucocytes, which is accompanied by an increased propensity for apoptosis, raised TNF-a and its soluble Fas ligand apoptosis receptor (sFasL) [27]. Probably the decreased levels of TNF-a would contribute to lower lymphocytes apoptosis, since this cytokine has a key role in apoptosis induction by the link to the sFasL receptor [3, 7]. IL-2 exerts regulatory effects in many body cells, being produced primarily by T and NK lymphocytes. This cytokine has a key role in humoral and inflammatory cellular responses [14]. In young mice submitted to 8 weeks of treadmill training, there were no significant changes in IL-2 production [10]. Pahlavani et al. [22] observed no alterations for IL-2 in rats submitted to swimming training in comparison with sedentary animals. These results are similar to the findings of the present study, since no significant alteration was observed for IL-2 after brief exercise at low or moderate intensity. Based on the results of the present study, one may conclude that short-term, low- and moderate-intensity exercise may be interesting for starting a physical activity program for sedentary individuals, since no deleterious alterations were observed in lymphocytes function. Exercise for a short time does not induce an increase in IL-6. Even so, greater increases would be detected on exercising for longer, in which IL-6 would play a determinant role in metabolism or an inflammatory response. Ferreira et al. [3] observed a decrease in plasma levels of TNF-a after 5 and 15 minutes of moderate-intensity exercise in rats. However, there have been reports of an increase in the level of TNF-a after exercise [6, 21]. Petersen and Pedersen [24] proposed that exercise primarily causes the release of IL-6, followed by increases in IL-1ra, IL-10 (anti-inflammatory cytokines) and soluble TNF-a receptor (sTNF-R), inhibiting TNF-a production. The findings described above showed that the stability or decrease in plasma levels of TNF-a could be related to the results found in the present study. Although this study found no significant changes in IL-6, a decrease in TNF-a was detected for groups exercised at moderate intensity. Exercise intensity played a role in alterations of TNFa, due to the greater decrease observed at moderate intensity for 15 minutes in comparison with low intensity for the same length of time.

Prestes J et al. Lymphocyte and Cytokines Int J Sports Med 2008; 29: 1010 1014

Downloaded by: Dot. Lib Information. Copyrighted material.

1014

Immunology

In summary, exercise performed for brief periods (5 and 15 minutes) can promote acute alterations in the immune system, inducing leukocytosis, increase in the number of circulating and tissue lymphocytes, decrease in DNA fragmentation in circulating lymphocytes and reduction in plasma levels of TNF-a. The present study showed that exercise performed for a short duration at low and moderate intensity is not innocuous with regard to the immune system response. The sum of acute responses observed in this study may exert a protective effect against sickness and may be used to improve health and lifespan [19].

References
1 Benoni G, Bellavite P, Adami A, Chirumbolo S, Lippi G, Brocco G, Giulini GM, Cuzzolin L. Changes in several neutrophil functions in basketball players before, during and after the sports season. Int J Sports Med 1995; 16: 34 37 2 Concordet JP, Ferry A. Physiological programmed cell death in thymocytes is induced by physical stress (exercise). Am J Physiol 1993; 265: C626 C629 3 Ferreira CKO, Prestes J, Frollini AB, Donatto FF, Dias R, Guereschi MG, Brambilla SR, Boaventura MFC, Pithon-Curi TC, Curi R, Verlengia R, Palanch AC, Cavaglieri CR. Influence of short duration acute exercise on the number, viability, functionality and apoptosis of neutrophils in sedentary rats. J Exerc Physiol Online 2007; 10: 27 36 4 Gabriel HH, Kindermann W. Adhesion molecules during immune response to exercise. Can J Physiol Pharmacol 1998; 76: 512 523 5 Goebel MU, Mills PJ. Acute psychological stress and exercise and changes in peripheral leukocyte adhesion molecule expression and density. Psychos Med 2000; 62: 664 670 6 Goebel MU, Mills PJ, Irwin MR, Ziegler MG. Interleukin-6 and tumor necrosis factor-a production after acute psychological stress, exercise, and infused isoproterenol: differential effects and pathways. Psychos Med 2000; 62: 591 598 7 Green KJ, Rowbottom DG. Exercise-induced changes to in vitro T-lymphocyte mitogen responses using CFSE. J Appl Physiol 2003; 95: 57 63 8 Hoffman-Goetz L, Zajchowski S, Aldred A. Impact of treadmill exercise on early apoptotic cells in mouse thymus and spleen. Life Sci 1999; 64: 191 200 9 Host CR, Norton KI, Olds TS, Lowe EL, Mulligan SP. The effects of altered exercise distribution on lymphocyte subpopulations. Eur J Appl Physiol 1995; 72: 157 164 10 Kohut ML, Thompson JR, Lee W, Cunnick JE. Exercise training-induced adaptations of immune response are mediated by b-adrenergic receptors in aged but not young mice. J Appl Physiol 2004; 96: 1312 1322 11 Kroemer G, Dallaporta B, Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Ann Rev Physiol 1998; 60: 619 642

Prestes J et al. Lymphocyte and Cytokines Int J Sports Med 2008; 29: 1010 1014

Downloaded by: Dot. Lib Information. Copyrighted material.

12 Lagranha CJ, Senna SM, De Lima TM, Silva EPP, Doi SQ, Curi R, PithonCuri TC. Beneficial effect of glutamine on exercise-induced apoptosis of rat neutrophils. Med Sci Sports Exerc 2004; 36: 210 227 13 Mazzeo RS. Catecholamine responses to acute and chronic exercise. Med Sci Sports Exerc 1991; 23: 839 845 14 McFarlin BK, Flynn MG, Stewart LK, Timmerman KL. Carbohydrate intake during endurance exercise increases natural killer cell responsiveness to IL-2. J Appl Physiol 2004; 96: 271 275 15 Moldoveanu AI, Shepard RJ, Shek PN. Exercise elevates plasma levels but not gene expression of IL-1 beta, IL-6, and TNF-alpha in blood mononuclear cells. J Appl Physiol 2000; 89: 1499 1504 16 Nemet D, Rose-Gottron CM, Mills PJ, Cooper DM. Effect of water polo practice on cytokines, growth mediators, and leukocytes in girls. Med Sci Sports Exerc 2003; 35: 356 363 17 Nielsen HB, Secher NH, Kristensen JH, Christensen NJ, Espersen K, Pedersen BK. Splenectomy impairs lymphocytosis during maximal exercise. Am J Physiol 1997; 272: R1847 R1852 18 Nieman DC. Exercise, upper respiratory tract infection, and the immune system. Med Sci Sports Exerc 1994; 26: 128 139 19 Nieman DC, Pedersen BK. Exercise and immune function: recent developments. Sports Med 1999; 27: 73 80 20 Opferman JT, Korsmeyer SJ. Apoptosis in the development and maintenance of the immune system. Nature Immunol 2003; 4: 410 415 21 Ostrowski K, Rohde T, Asp S, Schjerling P, Pedersen BK. Pro- and anti-inflammatory cytokine balance in strenuous exercise in humans. J Physiol 1999; 515: 287 291 22 Pahlavani MA, Cheung TH, Chesky JA, Richardson A. Influence of exercise on the immune function of rats of various ages. J Appl Physiol 1988; 64: 1997 2001 23 Pedersen BK, Hoffman-Goetz L. Exercise and the immune system: regulation, integration, and adaptation. Physiol Rev 2000; 80: 1055 1081 24 Petersen AMW, Pedersen BK. The anti-inflammatory effect of exercise. J Appl Physiol 2005; 98: 1154 1162 25 Quadrilatero J, Hoffman-Goetz L. N-acetyl-L-cysteine prevents exercise-induced intestinal lymphocyte apoptosis by maintaining intracellular glutathione levels and reducing mitochondrial membrane depolarization. Biochem Biophys Res Com 2004; 319: 894 901 26 Quadrilatero J, Hoffman-Goetz L. N-acetyl-L-cysteine inhibits exercise induced lymphocyte apoptotic protein alterations. Med Sci Sports Exerc 2005; 37: 53 56 27 Tuan TC, Hsu TG, Fong MC, Hsu CF, Tsai KKC, Lee CY, Kong CW. Deleterious effects of short-term, high-intensity exercise on immune function: evidence from leucocyte mitochondrial alterations and apoptosis. Br J Sports Med 2008; 42: 11 15 28 Voltarelli FA, Gobatto CA, Mello MAR. Determination of anaerobic threshold in rats using the lactate minimum test. Braz J Med Biol Res 2002; 35: 1389 1394