Effects of Partial Sleep Deprivation on

Chronobiology International, Early Online: 1–8, (2012) Copyright © Informa Healthcare USA, Inc. ISSN 0742-0528 print/1525-6073 online DOI: 10.3109/07420528.2012.742102

Effects of Partial Sleep Deprivation on Proinflammatory Cytokines, Growth Hormone, and Steroid Hormone Concentrations During Repeated Brief Sprint Interval Exercise Salma Abedelmalek,1 Nizar Souissi,2 Hamdi Chtourou,2,6 Meriam Denguezli,1 Chirine Aouichaoui,1 Monia Ajina,1 Asma Aloui,2 Mohamed Dogui,3 Samy Haddouk,4,5 and Zouhair Tabka1 Department of Physiology, Sousse Faculty of Medicine, Sousse, Tunisia, 2Research Laboratory “Sports Performance Optimization,” National Center of Medicine and Science in Sports (CNMSS), Tunis, Tunisia, 3Research Unit “Neurophysiology of Vigilance, of Attention and Performances,” 99/UR/08-23, Service of Functional Exploration of the Nervous System, University hospital Sahloul, Sousse, Tunisia, 4Department of Immunology, Habib Bourguiba University Hospital of Sfax, Sfax, Tunisia, 5 Faculty of Medicine of Sfax, Sfax University, Sfax, Tunisia, 6Research Unit EM2S, High Institute of Sport and Physical Education, Sfax University, Sfax, Tunisia

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The purpose of this study was to evaluate the effects of partial sleep deprivation (PSD) on circulating concentrations of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in relation to the secretory profiles of growth hormone (GH), cortisol, and testosterone during a repeated brief sprint interval exercise. Thirty healthy football players (mean age: 21.1 [range: 18–24] years; body mass index [BMI]: 22.6 [range: 18.47–24.46] Kg/m2) completed two test sessions at 08:00 h, one scheduled after a baseline night (bedtime: from 22:30 to 07:00 h) and the other after a PSD night caused by an early awakening (bedtime: from 22:30 to 03:00 h). During each session, participants performed 4 × 250-m run on a treadmill at a constant intensity of 80% of the personal maximal speed with a 3-min recovery in between. Tests session were performed at 08:00 h. Blood samples were collected before, immediately after the first and the fourth 250-m run, and 60 min after the exercise. The results showed that cortisol concentrations were not affected by the PSD. However, GH and testosterone concentrations were higher ( p < .05) 60 min after the exercise during PSD in comparison with baseline. Likewise, plasma concentrations of IL-6 and TNF-α were higher ( p < .05) after PSD during the exercise (i.e., the first and the fourth run) and remained elevated during the recovery period (i.e., 60 min after the exercise). In conclusion, these results showed that sleep restriction increases the proinflammatory cytokine, GH, and testosterone concentrations after physical exercise but did not affect the cortisol responses. (Author correspondence: [email protected]) Keywords: Anaerobic exercise, Cortisol, Growth hormone, IL-6, Sleep deprivation, Sprint, Testosterone, TNF-α

INTRODUCTION

loss results in increased sleepiness and decrements in neurobehavioral as well as physical performance (Abedelmalek et al., 2012; Dorrian et al., 2006; Persson et al., 2006; Reilly & Edwards, 2006; Souissi et al., 2003, 2008). Concerning the effect of sleep deprivation on the endocrine and immune system, it has been demonstrated that sleep loss is associated with next-day increase in proinflammatory cytokines (e.g., interleukin-6 [IL-6] and tumor necrosis factor-alpha [TNF-α]), which have been proposed as mediators of pathological or experimentally induced sleepiness in humans. Moreover, the increase of proinflammatory cytokines has been shown to be associated with an unfavorable metabolic profile

Sleep is commonly considered as a restorative process that influences the homoeostatic regulation of the neuroendocrine and immune systems (Dinges, 1995). Although both athletes and coaches believe that adequate sleep is essential for peak performance (Chtourou & Souissi, 2012; Souissi et al., 2012a, 2012b), there are many situations in which sleep is disturbed prior to an athletic event. Indeed, sleep loss, either total or partial, may be experienced by athletes who have to get up early in the morning to travel to a competition or who cannot fall asleep because of the psychological stress of a major event (Hall et al., 1998). In this context, sleep

Submitted December 19, 2011, Returned for revision January 23, 2012, Accepted October 17, 2012 Address correspondence to Dr. Nizar Souissi, Laboratoire de recherche “Optimisation de la performance sportive,” Centre National de la Médecine et des Sciences du Sport, Bp263, Ave Med Ali Akid, 1004 ElMenzah, Tunis, Tunisia. Tel.: +216 96 81 86 33; E-mail: [email protected]

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 S. Abedelmalek et al. (Vgontzas et al., 1999, 2004, 2005). Likewise, Irwin et al. (2006) showed that IL-6 and TNF-α increased significantly at 08:00 h after one night of partial sleep deprivation (PSD). Thus, IL-6 (i.e., a proinflammatory cytokine that is secreted by macrophages in response to infectious challenge) is a pleiotropic cytokine and its secretion is stimulated by physiological, psychological, and pathological stressors (Robson, 2003). In addition, IL-6 is a potent stimulator of the hypothalamic-pituitary-adrenal axis, stimulating the adrenocorticotropic and cortisol (Späth-Schwalbe, 1998) as well as growth hormone (GH) (Papanicolaou et al., 1998) secretions. A disturbed sleep might have an effect on the sleepwake cycle and the nocturnal secretion of GH and cortisol (Vgontzas et al., 2000, 2005). In fact, GH and cortisol secretions are closely related to the sleep-wake cycle (Spiegel et al., 1994, 1995). The release of GH occurs during the first period of sleep (i.e. non–rapid eye movement), whereas cortisol secretion is low during the first hours of sleep, but increases dramatically during early morning when the individual is still asleep (Kupfer et al., 1983). However, the secretion of inflammatory cytokines (i.e., IL-6, TNF-α) as well as GH and cortisol does not depend only on sleep. These hormones and cytokines are often secreted in response to physical stress (Mougin et al., 2001; Pedersen et al., 2001, 2003) Regarding the effect of sleep deprivation on hormonal responses during exercise, Opstad (1991) showed that sleep deprivation did not affect the plasma hormone concentrations in response to bicycle exercise during prolonged strain in the morning. In addition, Martin et al. (1986) found no change in cortisol concentrations during 3 h of mild exercise after prolonged sleep loss, or during 30 min at 65% of the maximal oxygen uptake after two nights of fragmented sleep. However, Mougin et al. (2001) showed alterations in the hormonal responses to submaximal exercise after one night of PSD. Indeed, the concentrations of cortisol were lower at 30 min post-exercise after PSD (i.e., at the beginning or at the end of the night). Recently, Nindl et al. (2006) showed that intense physical activity superimposed on energy and sleep restriction induced amplifications in GH and luteinizing hormone (LH) secretion (no change in pulse number) as well as decreases in hormone releases that these signaling peptides are responsible for stimulating, namely insulin-like growth factor-I (IGF-I) and testosterone. Exercise can also induce changes in immunological parameters (Nieman et al., 2001, 2003, 2007; Shephard & Shek, 1999). In this context, Nieman (1998) suggest that moderate-intensity exercise stimulates the immune system and may be somewhat responsible for exerciserelated reduction in illness. However, strenuous highintensity exercise induces immune suppression and may explain the increased risk of infection in athletes (Pedersen & Hoffman-Goetz, 2000). Recently, Meckel et al. (2009) showed that a brief sprint interval exercise

(4 × 250-m run) may lead to different anabolic/catabolic and inflammatory responses. The authors showed that maximal exercise was associated with significant increase in GH, testosterone, and the testosterone/cortisol ratio. In addition, exercise led to a significant increase in the plasma IL-6 that may indicate its important role in muscle tissue repair. Thus, exercise results in strong inflammatory responses. More recently, Meckel et al. (2011) showed that two types of sprint interval sessions (i.e., increasing and decreasing distance [400, 300, 200, 100 m]) led to a significant increase in the circulating pro- and anti-inflammatory mediators (i.e., IL-1, IL-6, and IL1ra) as well as GH, IGF-I, and testosterone levels. The authors also showed that IL-6 remained elevated in both sessions after 1 h of recovery. However, to date, the effect of PSD and exercise on immunological responses is largely unknown. It is critical, therefore, for athletes interested in maximal performance, as well as coaches and researchers, to examine the impact of PSD on the anabolic and the inflammatory status of the muscle during a repeated high-intensity exercise. Indeed, intermittent high-intensity exercise with a relatively short time intervals are deemed relevant fitness prerequisites in football. Thus, the aim of this study was to investigate the effects of PSD scheduled at the end of the night on plasma concentrations of IL-6, TNF-α, GH, testosterone, and cortisol during a repeated sprint exercise (4 × 250-m run at 80% of the maximal speed) in football players. In light of the literature observations, we hypothesized that PSD affects the proinflammatory cytokine, GH, and steroid hormone responses to repeated high-intensity exercises. METHODS Subjects Thirteen healthy football players (age: 21.1 [range: 19–24] years; height: 178.5 [range: 173.4–182] cm; body mass: 72.1 [range: 61.2–79.9] kg; BMI: 22.6 [range: 18.47– 24.46]) participated in the study. They were given a detailed explanation of the protocol before signing an informed consent form. The exact inclusion/exclusion criteria were that (i) they were trained subjects (i.e., football players); (ii) they kept standard times for sleeping habits (sleeping between 23:00 and 07:00 ± 1:00 h) and for eating prior to the beginning of the study (breakfast at 07:00 ± 1:00 h, lunch at 12:00 ± 1:00 h, and dinner at 20:00 ± 1:00 h); (iii) they were nonsmokers and did not consume caffeine or alcoholic beverages; and (iv) they were selected as “neither type” on the basis of their answers to the Horne and Ösberg self-assessment questionnaire (Horne & Östberg, 1976). The study protocol was in accordance with the Helsinki declaration for human experimentation and was approved by the University Ethics Committee. It also complied with the ethical and procedural requirements of the journal for the conduct of human biological rhythm research (Portaluppi et al., 2010). Chronobiology International


Sleep Deprivation and Exercise  Experimental Design All participants were regularly sleeping between 22:30 h and 07:00 h. In order to get accustomed with the change of bed (home vs. laboratory), they slept for four consecutive nights in the laboratory before the commencement of the assessments. Then, they completed two night sleep conditions. The first was a baseline night during which subjects were synchronized with a nocturnal rest from 22:30 to 07:00 h. The second night was a PSD condition during which they went to bed at 22:30 h and were woken up at 03:00 h. They were not allowed to sleep thereafter. Participants were kept awake by passive means such us watching TV and were prohibited from taking any food or drink any known stimulant (e.g., caffeine). In addition, they were continuously controlled by a technician. After each sleep condition, subjects performed a brief sprint interval exercise at 08:00 h. Body weight was measured to the nearest .1 kg using a Tanita digital scale (Tanita, Tokyo, Japan). Spontaneous body movement was assessed continuously by wrist actigraphy (Actiwatch Sleep & Activity software, version 5.32; Cambridge Neurotechnology, Cambridge, UK). Each actigraph contained a piezoelectric transducer sensitive to movements of .1-g acceleration. Actimetric devices were worn on the nondominant arm from 20:00 h the day before each test session to the end of the experiment. The actigraphs were returned to the laboratory and the data downloaded from the memory into a personal computer via an actigraph interface unit. Time series were first edited using information listed in the diaries to remove segments during which the monitor was not worn. Total sleep duration decreased significantly from the control (07:30 ± 00:30 h) to the PSD (03:30 ± 00:20 h) night. Before the morning test sessions, only one glass (150 to 200 ml) of water was allowed, to avoid postprandial thermogenesis effects. After the morning sessions, unrestricted food intake was allowed. Before the evening test sessions, participants had the same standard isocaloric meal at 12:00 h. After that meal, only water was allowed ad libitum. The overall daily energy intake goal was set at 10.5 MJ (2500 kcal) per capita/day. Exercise Protocol The football players performed two maximal outdoor 100-m run with a 15-min recovery period in between. The best result was used to calculate every player’s maximal speed to determine the exercise intensity. This exercise consisted of 4 × 250-m run on a treadmill (motor-driven treadmill; COSMED T 170, Rome, Italy) at a constant intensity of 80% of the calculated maximal speed (i.e., from the maximal speed of the 100-m run), with 3 min of rest in between. Blood Samples and Analyses Blood samples were collected using an indwelling venous catheter before the exercise (after 15 min of rest), after the first and the fourth 250-m run, and 60 min after the © Informa Healthcare USA, Inc.

exercise. Then, serum tubes were centrifuged at 3000 rpm for 20 min at room temperature and stored at −80° C until the measurement of hormonal and immune parameters. The radioimmunoassay was measured in triplicate for each sample. Testosterone and cortisol concentrations were determined by a commercial radioimmunoassay and GH concentrations were evaluated using IRMA kits (Immunotech, Marseille, France). The intra- and interassay coefficients of variation (CVs) were 1.5% and 13%, 8.9% and 2.8%, and 2.8 % and 5.3% for the GH, testosterone, and cortisol, respectively. The limit detections were .1, .1, and 7 ng·mL−1 for GH, testosterone, and cortisol, respectively. IL-6 and TNF-α were analyzed by enzyme-linked immunosorbent assay (ELISA) with the using a commercial kit (Immunotech). The intra-assay CVs were 1.6–6.8% and 1.6–10% and the interassay CVs were 7.9–14.6% and 5.4– 12.8% for IL-6 and TNF-α, respectively. Prior to statistical analyses, all data were corrected for changes in plasma volume using the method of Costill and Fink (1974). All assays were carried out as advised by manufacturer’s directions. To eliminate interassay variance, every player’s samples were assayed in the same assay. Statistical Analyses Statistical tests were processed using STATISTICA Software (StatSoft, xx, France). Data are reported as mean ± SD. Once the assumption of normality was confirmed using the Shapiro-Wilk W test, parametric tests were performed. Hormonal and inflammatory mediators’ data were analyzed using a two-way analysis of variance (ANOVA) (2 [sleep condition] × 4 [points of measurement]). When the ANOVA indicated significant sleep condition or points of measurement effects or a significant interaction sleep condition × points of measurement, significant differences between means were tested using the least significant difference (LSD) post hoc test. The level of statistical significance was set at p < .05.

RESULTS Hormone Concentrations Exercise was associated with an increase in GH (F(3, 36) = 4.69; p < .01) immediately after the first and the fourth run. However, testosterone concentrations increased only after the fourth run (F(3, 36) = 4.97; p < .01) (Figure 1). However, there was no significant effects of exercise on cortisol concentrations (F(3, 36) = 1.38; p < .05) (Figure 1). The sleep condition × points of measurement interaction for GH (F(3, 36) = 69.93; p < .001) and testosterone (F(3, 36) = 7.22; p < .001) concentrations were significant, indicating increases in these parameters after PSD ( p < .01) in comparison with baseline. However, the sleep condition × points of measurement interaction


 S. Abedelmalek et al.

FIGURE 1. Growth hormone (GH) (A), testosterone (B), and cortisol (C) concentrations (ng·mL−1) measured before the exercise (pre-run), after the first (run 1) and the fourth (run 4) run, and after the exercise (post-run) during baseline and partial sleep deprivation (PSD) conditions. All values are expressed as mean (± SD). *Significant difference with respect to pre-run during the same condition ( p < .05); ¥ significant difference in comparison with baseline (p < .05). Chronobiology International


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FIGURE 2. Plasma concentrations of interleukin-6 (IL-6) and TNF-α measured before the exercise (pre-run), after the first (run 1) and the fourth (run 4) run, and after the exercise (post-run) during baseline and partial sleep deprivation (PSD) conditions. All values are expressed as mean (± SD). *Significant difference with respect to pre-run ( p < .01) during the same condition; ¥significant difference in comparison with baseline ( p < .05).

for cortisol (F(1, 12) = .24; p > .05) and the sleep condition effect for all hormones were not significant ( p > .05).

Inflammatory Mediators The effects of PSD on inflammatory mediators during the exercise are summarized in Figure 2. Both PSD and baseline conditions were associated with increases in IL-6 (F(3, 36) = 28.29; p < .001) and TNF-α (F(3, 36) = 9.28; p < .01) during the exercise (i.e., immediately after the first and the fourth run). The sleep condition effect on the circulating IL-6 (F(1, 12) = 4.11; p > .05) and TNF-α (F(1, 12) = .15; p > .05) was not significant. The sleep condition × points of measurement interaction for IL-6 (F(3, 36) = 21.58; p < .001) and TNF-α (F(3, 36) = 34.02; p < .01) was significant, indicating that the effect of PSD is more pronounced 60 min © Informa Healthcare USA, Inc.

after exercise in comparison with the other points of measurements (Figure 2). Indeed, the increase in plasma concentrations of IL-6 and TNF-α is significantly higher 60 min after exercise in comparison with the other points of measurements during PSD than baseline (p < .01).

DISCUSSION The results of this study showed that PSD timed at the end of the night leads to alterations in immunological responses during a repeated high-intensity exercise. Indeed, IL-6, TNF-α, GH, and testosterone were higher 60 min after the brief sprint interval exercise during PSD compared with the baseline night. However, cortisol concentrations were unaffected by PSD.


 S. Abedelmalek et al. Our results showed that PSD didn’t affect the cortisol concentrations in response to the anaerobic exercise. This finding confirms those of Martin et al. (1986) who found no change in cortisol concentrations during 3 h of mild exercise after prolonged sleep loss, or during 30 min of exercise after two nights of sleep deprivation. Also, our results demonstrated that cortisol concentrations were unaffected by the sprint interval exercise. It is known that the exercise-induced cortisol increase depends on the duration and intensity of the physical activity. In this context, Urhausen et al. (2000) showed a significant increase in cortisol after an aerobic exercise (i.e., 60% of Vo2max). Therefore, it is possible that the exercise duration in the present study was not long enough to cause an increase in cortisol concentrations. Moreover, the sprint interval exercise was associated with significant increases in testosterone in the two sleep conditions, which could enhance the anabolic status in the muscle. These results agree with previous findings (Axelsson et al., 2005; Opstad, 1992). Indeed, Opstad (1992) found a 47% decrease in morning testosterone concentration after a 5-day military training course. Likewise, Opstad (1991) showed that sleep deprivation did not affect the hormonal responses to bicycle exercise during prolonged strain in the morning. However, previous researchers found that PSD may alter some physiological responses to subsequent exercise (Mougin et al., 1991). Nindl et al. (2001) demonstrated that the decline in overnight testosterone concentrations after acute heavy-resistance exercise is accompanied by a blunted LH production rate and elevated cortisol concentrations. Recently, Nindl et al. (2006) observed that brief periods of intensive physical activity superimposed on energy and sleep restriction induced amplifications in GH and LH secretions. However, due to methodological differences such as exercise type (e.g., intensity, duration, etc.) or sleep loss protocol, the results of the present study cannot be compared with those of previous investigations. For instance, Martin et al. (1986) and Opstad (1991) investigated the effect of prolonged (i.e., severe) sleep loss on hormones concentrations, whereas in the present study, the sleep loss was of short duration. Furthermore, the physical exercises used in the studies of Nindl et al. (e.g., heavy-resistance exercise; Nindl et al., 2001) have not the same characteristics as our study protocol, which may, in part, explain the discrepancies between the findings. Our results showed that GH concentrations increased during and after the sprint interval exercise in the two sleep conditions. In addition, previous studies have examined the effects of endurance exercise on GH concentrations and suggested that this exercise type was sufficient to cause a considerable metabolic effect to stimulate GH secretion with minimal exercise duration of 10 min (Felsing et al., 1992). Interestingly, our results found that a brief anaerobic interval exercise led to a significant increase in GH concentrations.

Moreover, Brandenberger et al. (2000) demonstrated that modifications (i.e., blunted) in the normal sleeprelated GH pulse after sleep deprivation is compensated during the day. Consequently, the amount of GH secreted during a 24-h period is similar whether or not a person has slept during the night. Likewise, it has been demonstrated that when sleep onset is delayed, the pulse of GH release, which is associated with stages 3 and 4 of the sleep period, is also delayed (Sassin et al., 1969). However, nocturnal secretion of GH is decreased or eliminated in the absence of slow wave sleep (Karacan et al., 1971; Sassin et al., 1969). Also, Steiger et al. (1987) suggested that resting in bed during the late evening hours or waiting for permission to sleep may be sufficient to trigger the release of GH. Immune parameters vary under different conditions, such as exercise, sleep, circadian rhythmicity, and stress (Irwin et al., 2006; Meckel et al., 2009). To the best of our knowledge, the present study is the first to assess the immune responses caused by brief sprint interval exercise after one night of PSD. Our results showed that plasma concentrations of IL-6 and TNF-α increased significantly during the exercise and remained elevated during the recovery period (i.e., 60 min after the exercise) after PSD. Consistently, previous reports showed that sleep deprivation was associated with next-day increase of proinflammatory cytokines, e.g., IL-6 and TNF-α (Harris et al., 1999; Shearer et al., 2001), which have been proposed as mediators of pathological or experimentally induced sleepiness in humans and have been shown to be associated with an unfavorable metabolic profile with implications for inflammatory and cardiovascular disease risk (Musselman et al., 1998; Papanicolaou et al., 1998). Also, Vgontzas et al. (2004) showed that sleep restriction by 2 h for 1 week in human is associated with increased daytime IL-6 levels, increased sleepiness, and decreased performance. Furthermore, exercise induces a general stress response involving the activation of the immune systems and may induce immunosuppression in the recovery period and continues to increase after exercise (Meckel et al., 2009, 2011). Additionally, the concentrations of TNF-α have been shown to increase 2–3-fold after exercise (Ostrowski et al., 1999). The major source for the exercise-related IL-6 increase is the skeletal muscle (Green et al., 2006). Indeed, previous studies clearly demonstrate that muscle contractions without any muscle damage induce a marked elevation in plasma IL-6 (Pedersen et al., 2001, 2003). However, IL-6 is believed to play an important mediatory role in the inflammatory response needed for the exercise-associated muscle damage repair (Steensberg et al., 2002). As PSD is associated with an increase in IL-6 and TNF-α concentrations the next day, the oversecretion of IL-6 and TNF-α during the recovery period observed in the present study could be explained by an association between the PSD and exercise. Another explanation suggested that glycogen depletion, a major stimulus for Chronobiology International

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IL-6 release (Miki et al., 1999), caused by muscle damage was a further stimulus for the elevation of IL-6 during the recovery phase. Furthermore, elevated plasma concentrations of IL-6 have been associated with the decrease of athletic performance and, therefore, increased levels of fatigue (Robson-Ansley et al., 2004). Study Limitations To the best of our knowledge, this is the first study that explores the effect of PSD on immunological and hormonal parameters changes during a repeated brief sprint interval exercise. The present study has some limitations. First, the participants were well-trained males and it remains unclear whether the results can be applied to sedentary subjects. Second, it would be better to examine the acute impact of resistance training program on immunological and hormonal parameters during exercise and recovery after PSD. Third, because of the limited sample amounts, other important inflammatory (e.g., IL-1) and hormonal (e.g., IGF-I, IGFbinding proteins [IGFBPs]) markers were not examined. Another limitation is related to the sleep amount. Indeed, subjects took different durations to fall asleep before the different conditions. CONCLUSION The findings of the current study showed that PSD increases the proinflammatory cytokine, GH, and testosterone concentrations after repeated bouts of high-intensity maximal exercise but did not affect the cortisol responses. As a result, the immunological profile in response to PSD and exercise can be monitored by the athlete, the coach, and the supporting staff and may be used as an objective tool to monitor the training load and to better plan training cycles throughout the training season and competition period. Furthermore, results from the field of exercise immunology may help to guide athletes and contribute to public health recommendations on exercise and infections. ACKNOWLEDGMENTS The authors wish to express their sincere gratitude to all participants for their maximal effort and cooperation. Declaration of Interest: This study was financially supported by the Ministry of Higher Teaching and Scientific Research, Tunisia. The authors report no conflict of interest. The authors alone are responsible for the content and writing of the article. REFERENCES Abedelmalek S, Chtourou H, Aloui A, Aouichaoui C, Souissi N, Tabka Z. (2012). Effect of time of day and partial sleep deprivation on © Informa Healthcare USA, Inc.

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