J Endocrinol Invest DOI 10.1007/s40618-014-0144-z
ORIGINAL ARTICLE
Trained and untrained males show reliable salivary testosterone responses to a physical stimulus, but not a psychological stimulus B. T. Crewther • L. P. Kilduff • C. J. Cook
Received: 25 May 2014 / Accepted: 26 July 2014 Ó Italian Society of Endocrinology (SIE) 2014
Abstract Background The testosterone (T) responses to a physical stimulus are thought to be more stable and reproducible compared to a psychological stimulus. Purpose This study compared the salivary T (Sal-T) responses to both stimuli in four groups of men: professional rugby players (n = 17), recreational rugby players (n = 10), a mixed athlete group (n = 14) and untrained controls (n = 12). Methods Each group completed three treatments: (1) watching a video with aggressive rugby footage, (2) performing a short bout of sprint exercise and (3) a control session. Saliva samples were taken before and 15 min after each treatment. Results The sprint exercise changes in Sal-T levels were similar in the elite rugby (17.1 ± 11.1 %), recreational rugby (11.9 ± 15.9 %), mixed athlete (27.6 ± 32.0 %) and control groups (25.3 ± 23.6 %). In response to the video, Sal-T increased in the elite rugby (6.9 ± 6.4 %) and untrained groups (11.9 ± 13.5 %), but decreased in the recreational rugby players (-7.5 ± 11.0 %). The individual Sal-T responses to the sprints were also correlated (r = 0.69 to 0.82) with other treatment responses.
B. T. Crewther (&) C. J. Cook Hamlyn Centre for Robotic Surgery, Imperial College South Kensington Campus, London SW7 2AZ, UK e-mail: [email protected] B. T. Crewther C. J. Cook School of Sport Health and Exercise Science Bangor University, Bangor, Wales, UK L. P. Kilduff C. J. Cook A-STEM, College of Engineering, Swansea University, Swansea, UK
Conclusions Sprint exercise had a more consistent effect on Sal-T than a video with aggressive content and thus, could provide a reliable stimulus for increasing T availability in men with different training backgrounds. Individual Sal-T reactivity also appears to be somewhat stable across different treatments. These data provide further understanding around the induction, moderation and interpretation of T physiology. Keywords Behaviour Athletes Neuroendocrine Hormones Saliva Video
Introduction Traditionally, the training role of testosterone (T) has been presented as being anabolic and anti-catabolic to protein synthesis and degradation, respectively [1]. This view has been challenged by work demonstrating that physiological elevations in T and other anabolic markers may not influence protein metabolism [2, 3]. Nevertheless, T might help to regulate other neuromuscular outputs (e.g. behaviour, motor system outputs, cognition, calcium signalling) linked to training performance and adaptation [4]. Thus, there is considerable interest in developing practical strategies to acutely modify T and these outcomes [4]. A psychological stimulus in the form of video clips provides an effective tool for acutely modifying blood T (total) or salivary T (Sal-T) levels in male populations [5– 7]. In a sporting context, watching a previous victory dramatically increased Sal-T secretion (by 42–44 %) in male hockey players, whereas watching a defeat or a neutral control video did not produce any change [8]. In professional male rugby players, small (4–10 %) but significant improvements in Sal-T levels were also observed
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with the presentation of short (4 min) video clips with different contents: motivational, aggressive, humorous and erotic [9]. These hormonal changes also mapped onto subsequent improvements in physical performance [9]. It does appear that the Sal-T response to a video clip is individual in nature and possibly sport and elite specific [9]. As an example, watching a video with somewhat aggressive content (e.g. big rugby tackles) produced the greatest Sal-T response in elite male rugby players [9]. However, the same video footage failed to reproduce a significant hormonal change in healthy men [10], whereas a documentary film showing real-life aggression and violence did elevate Sal-T [7]. Thus, the situational context and emotional importance of the video footage to the assessed population may dictate whether a T response will occur. To our knowledge, no studies have examined the effect of a video clip showing aggressive sporting actions in male populations who differ in training level and background. Physical exercise also provides an effective stimulus for increasing blood (free, total, bioavailable) T and Sal-T levels in men, including continuous running [11, 12], interval running [12, 13], sub-maximal and maximal cycling [14], and weight training [15, 16]. Gonadal steroids are particularly responsive to sprint exercise [17–21], possibly due to the induction of important mechanical and physiological signals. Sprint exercise is also free of other constraints related to a video display (e.g. situational context, emotional value) and thus, could provide a more reliable stimulus for elevating T in both trained and untrained groups. However, no studies have compared the Sal-T responses to a psychological (video) and physical (sprint exercise) stimulus in men with different training backgrounds. This study compared the effects of a video clip with aggressive content and sprint exercise on the Sal-T responses in four groups of men: elite rugby players, recreational rugby players, mixed athletes involved in noncontact sports, and untrained controls. We hypothesized that sprint exercise would induce greater and more consistent Sal-T changes (increases) than the video clip, and that the video (due to the rugby-related aggressive content) would promote greater Sal-T responses in the elite rugby and recreational rugby groups than the mixed athletes and untrained men. Based on prior work [22, 23], we also examined the stability of the individual T responses across the different treatments.
Materials and methods Participants The study population (n = 53) comprised elite rugby players (n = 17, 21.5 ± 1.0 years, 186 ± 6 cm and 99.2 ±
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8.2 kg), recreational rugby players (n = 10, 21.1 ± 2.1 years, 183 ± 5 cm and 89.7 ± 8.7 kg), a mixed group of athletes involved in non-contact sports (n = 14, 23.1 ± 5.4 years, 184 ± 8 cm and 88.5 ± 8.9 kg) and untrained controls (n = 12, 21.0 ± 1.5 years, 180 ± 4 cm and 84.8 ± 7.2 kg). The elite rugby players were full-time professional athletes, whereas the recreational rugby players were involved in a local rugby competition. The mixed athletes were participating in various sports (cricket, football, bodybuilding and rowing) at either a local or national level, whilst the controls were not involved in any organized sport. Pre-screening assessments indicated that the study participants had no known disorders (e.g. endocrine, psychiatric) and were not taking any medicines or drugs (e.g. anabolic steroids) over the last 12 months that could influence the study outcomes. All data were collected with University ethics approval and participants signed informed consent. Experimental design Data from several experimental trials were collated for this study. In all trials undertaken, the participants were assessed using a crossover design after random allocation to three treatments: (1) watching a short video clip with aggressive content, (2) completing a short bout of sprint exercise and (3) a control session with no stimuli. Saliva samples were taken before and after each stimulus, along with time-matched samples across the control session. The samples were assayed for Sal-T levels and examined for changes within and differences between the study groups and treatments. Testing protocols Each treatment described above was separated by at least 3 days (up to a maximum of 7 days) and the participants were told to refrain from performing any exercise in the morning before each test. In addition, all intense exercise (especially for the lower body) was avoided in the 48-h period before testing to eliminate the confounding effects of fatigue. The study groups were assessed in the afternoon (between 1200 and 1600 h) to address expected time-of-day variation in T secretion and reactivity to physical or psychological stressors [24–26]. Furthermore, each participant completed their own assessments at a similar time of day (±1 h). Before the sprint assessment (see below), a familiarization session was performed to ensure that the participants were capable of completing the exercise procedures and to account for a pronounced stress response occurring from intense, unaccustomed exercise.
J Endocrinol Invest
Sprint exercise testing
Control testing
The participants arrived at the laboratory and sat quietly for 10 min in a stress-free environment. The exercise sprints were performed on a cycle ergometer (Monark 824E, Sweden) after a short warm-up with a light load. Three intermittent cycling protocols were used (5 9 10 s, 4 9 10 s, 3 9 10 s) with resistance loads ranging from 5 to 7.5 % of individual body mass. Short recovery periods (e.g. 40–60 s) were provided between each sprint repetition. The entire protocol was completed in 3–5 min. The participants were led through the protocols by at least two researchers, who also timed each event, modified the test and recovery loads, and provided feedback on when to start and stop each sprint. The testing protocols and equipment ensured that the same muscle groups were activated over a similar time period, with a mean exercising duration of 40 s. Similar sprint cycling protocols have been shown to raise blood T or Sal-T levels in men [17, 18, 20, 27]. Strong verbal encouragement was provided to ensure that the sprints were performed with maximal effort. A short cool down was performed after the final sprint consisting of slow pedalling without load. The participants then sat quietly in the laboratory until sampling.
Control data were taken from participants after an initial 10-min resting period. Time-matched saliva samples (as per the sprint and video treatments) were taken under similar conditions. This involved either watching a blank video screen or simply sitting quietly in the testing facility for the allotted time period. The participants remained seated throughout to prevent any residual effects of physical exercise (e.g. standing up, walking around) on the hormonal milieu and they were tested as individuals or in small groups. Again, there were no interactions between the study investigators and participants across the control session except to provide verbal instructions prior to testing and sample collection.
Video clip testing The participants sat quietly for 10 min in the laboratory before watching a short (4–5 min) video showing aggressive content of big rugby tackles and related aggressive play [9, 10]. This type of video footage was previously shown to acutely elevate male Sal-T levels [9]. Three video clips were used across the various trials, although the aggressive rugby content remained the same. The clips were taken from an online resource and played for the allotted timeframe (http://www.youtube.com/watch?v= i0dH5-2Yqmw, http://www.youtube.com/watch?v=UAaxe TowTd0 and https://www.youtube.com/watch?v=UTg1Xm 9CJgo). During the video presentation, the participants either sat by themselves to watch the video on a computer screen or they sat in small groups (up to 4) to watch the video on a larger projector screen. Group testing was unavoidable due to time constraints and facility availability. There was no communication between the investigators and subjects during the video presentations, or in the short period after, to eliminate the effects of social interactions on circulating Sal-T levels [28]. The only interactions occurred when simple verbal instructions were provided before the video presentation. At the end of the video, the participants sat quietly in the laboratory before subsequent sampling.
Salivary hormone testing Saliva samples were collected before each stimulus (after the rest period) and 15 min after to coincide with expected T changes in this media [5, 9, 27, 29], which are typically measured at 10–15 min post stimulus. The timing between the pre and post saliva samples was approximately 20 min. Saliva (1–2 ml) was collected by passive drool (not using Salivettes) and without stimulation (e.g. citric acid) into sterile containers and stored at -80 °C until assay. To account for dietary factors, each participant was instructed to maintain the same food and fluid intake before each session, and to refrain from eating or drinking 2 h before sampling [30]. Although the hydration state of individuals may have varied due to the self-selection procedures, hydration state does not influence blood T before, during or after exercise [31]. After thawing and centrifugation, the samples were analysed for T using a commercial immunoassay (Salimetrics LLC, USA). The detection limit for each plate was 6.1 pg/mL with inter-assay CV’s of 5–12 %, based on low and high control samples. Intraassay CVs on duplicate samples are typically less than 4 %. Samples for each participant were tested in the same assay to eliminate inter-assay variance. Statistical analyses The Sal-T measures were log transformed before analysis to normalize data distribution and reduce non-uniformity bias. The raw data are presented to allow comparisons with other studies. To assess the Sal-T responses, the delta change scores were calculated across each treatment (pre to post change presented as a mean percent value) and compared with paired T tests. The change scores in Sal-T were examined using a two-way (group 9 treatment) analysis of variance with repeated measures on the treatment variable.
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J Endocrinol Invest Table 1 Salivary testosterone levels pre and post the video, sprint and control treatments in the different male groups (mean ± SD) Video
Elite rugby players
Sprint
Control
Pre
Post
Pre
Post
Pre
Post 146
M
141
151
124
145
146
SD
22
27
16
18
27
24
Recreational rugby players
M SD
139 41
130 44
147 35
164 39
138 31
142 31
Mixed athletes
M
125
129
115
150
128
124
Untrained controls
SD
23
27
19
49
38
29
M
139
161
150
192
154
150
SD
37
63
53
81
67
60
The unit of measurement is pg/mL
Where appropriate, post hoc testing was conducted using the Tukey–Kramer test for unbalanced groups. The stability of the individual Sal-T responses to each treatment was assessed using Pearson correlations. Significance was set at an alpha level of p B 0.05.
Results Group demographics We found no significant differences in the mean age (F(3,52) = 1.403, p = 0.252) or height (F(3,52) = 1.954, p = 0.133) of the four male groups, but they differed in body mass (F(3,52) = 8.259, p \ 0.001). The elite rugby players were 9.5, 10.7 and 14.3 kg heavier than the recreational rugby players, mixed athletes and untrained controls (p \ 0.01), respectively. Testosterone responses Table 1 shows the descriptive Sal-T results for each group across the different treatments. In terms of the Sal-T responses (Fig. 1), the elite group showed a positive change across the video (6.9 ± 6.4 %) and sprint (17.1 ± 11.1 %) treatments (p \ 0.001). Conversely, SalT decreased after the video (-7.5 ± 11.0 %, p = 0.043) and increased with exercise (11.9 ± 15.9 %, p = 0.039) in the non-elite rugby group. The mixed athletes also had a positive Sal-T response to the sprints (27.6 ± 32.0 %, p = 0.006). In the control group, both the video (11.9 ± 13.5 %) and sprint (25.3 ± 23.6 %) stimuli produced an elevated Sal-T response (p \ 0.012). No changes in Sal-T levels were seen across the control sessions in any group (p [ 0.415). Examination of the Sal-T changes revealed a significant treatment effect (F(2,98) = 27.268, p \ 0.001) and
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Fig. 1 Percent change in salivary testosterone (Sal-T) levels across the video, sprint and control treatments in the different male groups (mean ± SD). Significant change from baseline #p \ 0.05, * p \ 0.01. 1 Significant from all of the control sessions, 2 significant from the elite rugby, recreational rugby and mixed athlete group video sessions, 3 significant from the elite rugby, mixed athlete and control group control sessions, 4 significant from the recreational rugby group video session p \ 0.05
group 9 treatment interaction (F(6,98) = 2.277, p = 0.042). The Sal-T responses to the sprint sessions (elite rugby, mixed athlete and control groups) were significantly greater than most of the video and/or control results (p \ 0.05), with the sprint and video outcomes also differing in the recreational rugby group (p \ 0.05). Following the video clip, the control group had a greater Sal-T response than the recreational rugby players (p \ 0.05). The individual pre and post Sal-T data for each treatment are plotted in Fig. 2. Testosterone interrelationships In the recreational rugby group, the individual Sal-T changes across the sprint and control treatments were strongly and significantly related (Table 2). The Sal-T responses to the sprint and video treatments were also correlated in the mixed athlete and control groups.
Discussion The physical stimulus employed in this study produced reliable changes (increases) in Sal-T levels across all of the male groups tested, thereby demonstrating a consistent population effect. In some cases the psychological stimulus also promoted elevated Sal-T levels, but the observed changes were generally smaller than the physical stimulus and the Sal-T responses were group dependent, not reflected at the population level. As hypothesized, sprint exercise provided an effective stimulus for increasing Sal-T levels and the mean changes in the elite rugby (17.1 %), recreational rugby (11.9 %),
J Endocrinol Invest
Fig. 2 Individual salivary testosterone (Sal-T) levels pre and post the video, sprint and control treatments in the elite rugby (a), recreational rugby (b), mixed athlete (c) and control groups (d)
Table 2 Correlations between the changes in salivary testosterone levels across the video, sprint and control treatments in the different male groups Sprint vs. video
Sprint vs. control
Video vs. control
Elite rugby players
r = 0.19, p = 0.475
r = 0.09, p = 0.732
r = -0.05, p = 0.851
Recreational rugby players
r = -0.48, p = 0.165
r = 0.82, p = 0.004
r = -0.54, p = 0.120
Mixed athletes
r = 0.72, p = 0.004
r = 0.25, p = 0.383
r = 0.02, p = 0.955
Untrained controls
r = 0.69, p = 0.013
r = -0.08, p = 0.810
r = -0.32, p = 0.303
mixed athlete (27.6 %) and untrained control (25.3 %) groups were similar irrespective of sporting involvement, or lack thereof. Our results also confirm the general finding that high-intensity sprint exercise can rapidly increase blood T (free, total) levels, along with more active metabolites (e.g. dehydrotestosterone), and Sal-T levels in male populations with different training backgrounds [17– 21, 27, 29]. Whilst physical conditioning and repeated
exposure to training stress (habituation) may reduce the acute T response to exercise [15, 32], the sprint cycling test is based on a voluntary maximal effort and as such may normalize for these factors. This could explain similar SalT responses in all groups tested despite expected differences in exercise capacity and tolerance, cycling workloads, and motivational drive. In response to the video, Sal-T was elevated in the elite rugby (6.9 %) and control groups (11.9 %), and the magnitude of change was similar to previous work [9, 10]. It was surprising that the untrained men were closest to the professional rugby players, whilst the recreational rugby players showed a negative video response (-7.5 %). We expected some specificity within a sport due to the video content; however, professional rugby differs considerably in training and competition from recreational sport so our observations could reflect perceptual and emotional factors relating to playing status, as well as content affiliation with only elite rugby actions shown. Reports of declining Sal-T levels in men after viewing a sad [9] and stressful video [5] further suggest that emotive content and individual perception are potential factors regulating the T response to a visual presentation.
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Different video clips have been shown to modify Sal-T (up to 10 %) in a small but defined group of professional rugby players [9]. As we speculated, this effect was not reproducible across a broader group of athletes in this work. This may again reflect the emotional value that an athlete group ascribes to the video shown, for example, watching a video of prior wins elevated Sal-T (42–44 %) but viewing a loss had no effect [8]. Watching erotic or sexual films can promote similar changes (15–35 %) in male T levels [5, 6, 33], which could be linked to emotional bonding and sexual arousal. A rise in Sal-T after watching a violent film probably reflects a general increase in physiological arousal [7]. Extending prior work showing a link between these Sal-T changes and muscle performance [9], future research could address whether similar T surges (induced by different stimuli) can differ in their functionality, or whether different stimuli can be combined to augment this response. It appears that sprint exercise (vs. video clip) provides a stronger and more reliable stimulus for increasing T availability. Other research comparing an exercise and cognitive stressor in children support our findings [34]. Given that the psychological stress response is determined by complex cognitive-appraisal mechanisms, it is not surprising that the blood or salivary responses of T can vary with the presentation of this type of stimuli [26, 34–38]. This could be partly explained by individual variation in hormonal reactivity [26], as we observed in the group results (see Fig. 2). One study did report small but similar increases in Sal-T in men completing a sprint exercise trial and when playing a violent video game [27]. However, a video game presents a very different psychological stimulus with a level of immersion that is also highly competitive in nature (i.e. outcome dependant), rather than simply sitting and passively watching a video clip. Any measured change in T could be explained by mechanisms involving secretion and/or synthesis rates, systemic changes (e.g. plasma volume, blood flow and clearance rates, binding protein levels and dissociation), and depending on the collection media, tissue activity (e.g. steroid metabolism, conversion) [1, 12, 14, 29]. The T response to physical exercise can be attributed to mechanical (e.g. cadence), metabolic (e.g. lactate) and/or autonomic (e.g. adrenaline) factors [18, 19]. Psychological factors contributing to T secretion include stress perception [35, 37], verbal actions (e.g. talking) [37] and any verbal feedback received [39]. The T changes occurring with either physical or psychological stimulation are also linked to other stress outcomes (e.g. cortisol, adrenocorticotropic hormone) [14, 38, 40] and hormonal predisposition (e.g. pre-test T levels) [26]. The relationships between the Sal-T responses to sprint exercise and the other treatments are novel findings, which
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also appear to be group dependent. Similar results were seen in other athlete groups across training sessions [23], competitions [22], as well as practice sessions and competition [41]. Collectively, these data confirm that T reactivity is somewhat stable on an individual level, especially when anchored against one or more physical stressors. By default, these results also confirm consistent variation in the blood T or Sal-T responses of individual athletes when exposed to the same exercise stressors [16, 42], and again evident in Fig. 2, which could indicate a genetic ability to respond to, and cope with, a stress challenge [4]. Indeed, prescribing workouts based on individual T variation have been used successfully to maximize short-term training adaptations [43]. Overall, our results suggest that sprint exercise provides a more reliable stimulus for modifying (increasing) Sal-T availability across broad populations and that these measurements are themselves stable. Therefore, this form of exercise could be used to assess T rhythmicity and dynamics [18, 27], to profile individual stress adaptations [42, 43], and modify physical performance [20]. The video shown produced less stable results that were group specific and probably dependent on a large number of factors such as emotive meaning, training status and specificity of sport, as well as cultural factors with respect to the video content. This makes it less suitable as a model for general population studies, but still interesting for examining the emotive content of a video as a modifier of T, behaviour and/or muscle performance [5, 8, 9]. We do acknowledge some of the study limitations, such as slight differences in the exercising protocols and the unbalanced groups. Although the general content of the videos was consistent across the study groups, the actual footage did show different rugby teams and players, along with different aggressive actions that could also influence how individuals perceive and subsequently respond to this. Furthermore, we did not address subject habituation to the video footage shown, nor did we monitor participant exposure to other psychological stressors before testing. The lack of data on other biomarkers of stress (e.g. cortisol) and the gonadal axis (e.g. gonadotropin-releasing hormone, luteinizing hormone) is other consideration. Research that addresses these issues would add value to the fields of behavioural endocrinology, sport and exercise, and general stress physiology. In conclusion, a physical stimulus involving sprint exercise gave a more reliable population response for Sal-T (increasing) in trained and untrained men than a psychological stimulus involving a video clip with aggressive content (increasing, decreasing and no change). Individual Sal-T reactivity was correlated across different treatments to indicate a level of stability in these measurements.
J Endocrinol Invest Acknowledgments We thank the participants, researchers and trainers who contributed to this study. This project was supported by the Elite Sport Performance Research in Training with Pervasive Sensing Programme [EP/H009744/1], funded by the Engineering and Physical Sciences Research Council, and the UK Sports Council.
16.
17. Conflict of interest
The authors declared no conflict of interests. 18.
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ORIGINAL ARTICLE
Trained and untrained males show reliable salivary testosterone responses to a physical stimulus, but not a psychological stimulus B. T. Crewther • L. P. Kilduff • C. J. Cook
Received: 25 May 2014 / Accepted: 26 July 2014 Ó Italian Society of Endocrinology (SIE) 2014
Abstract Background The testosterone (T) responses to a physical stimulus are thought to be more stable and reproducible compared to a psychological stimulus. Purpose This study compared the salivary T (Sal-T) responses to both stimuli in four groups of men: professional rugby players (n = 17), recreational rugby players (n = 10), a mixed athlete group (n = 14) and untrained controls (n = 12). Methods Each group completed three treatments: (1) watching a video with aggressive rugby footage, (2) performing a short bout of sprint exercise and (3) a control session. Saliva samples were taken before and 15 min after each treatment. Results The sprint exercise changes in Sal-T levels were similar in the elite rugby (17.1 ± 11.1 %), recreational rugby (11.9 ± 15.9 %), mixed athlete (27.6 ± 32.0 %) and control groups (25.3 ± 23.6 %). In response to the video, Sal-T increased in the elite rugby (6.9 ± 6.4 %) and untrained groups (11.9 ± 13.5 %), but decreased in the recreational rugby players (-7.5 ± 11.0 %). The individual Sal-T responses to the sprints were also correlated (r = 0.69 to 0.82) with other treatment responses.
B. T. Crewther (&) C. J. Cook Hamlyn Centre for Robotic Surgery, Imperial College South Kensington Campus, London SW7 2AZ, UK e-mail: [email protected] B. T. Crewther C. J. Cook School of Sport Health and Exercise Science Bangor University, Bangor, Wales, UK L. P. Kilduff C. J. Cook A-STEM, College of Engineering, Swansea University, Swansea, UK
Conclusions Sprint exercise had a more consistent effect on Sal-T than a video with aggressive content and thus, could provide a reliable stimulus for increasing T availability in men with different training backgrounds. Individual Sal-T reactivity also appears to be somewhat stable across different treatments. These data provide further understanding around the induction, moderation and interpretation of T physiology. Keywords Behaviour Athletes Neuroendocrine Hormones Saliva Video
Introduction Traditionally, the training role of testosterone (T) has been presented as being anabolic and anti-catabolic to protein synthesis and degradation, respectively [1]. This view has been challenged by work demonstrating that physiological elevations in T and other anabolic markers may not influence protein metabolism [2, 3]. Nevertheless, T might help to regulate other neuromuscular outputs (e.g. behaviour, motor system outputs, cognition, calcium signalling) linked to training performance and adaptation [4]. Thus, there is considerable interest in developing practical strategies to acutely modify T and these outcomes [4]. A psychological stimulus in the form of video clips provides an effective tool for acutely modifying blood T (total) or salivary T (Sal-T) levels in male populations [5– 7]. In a sporting context, watching a previous victory dramatically increased Sal-T secretion (by 42–44 %) in male hockey players, whereas watching a defeat or a neutral control video did not produce any change [8]. In professional male rugby players, small (4–10 %) but significant improvements in Sal-T levels were also observed
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with the presentation of short (4 min) video clips with different contents: motivational, aggressive, humorous and erotic [9]. These hormonal changes also mapped onto subsequent improvements in physical performance [9]. It does appear that the Sal-T response to a video clip is individual in nature and possibly sport and elite specific [9]. As an example, watching a video with somewhat aggressive content (e.g. big rugby tackles) produced the greatest Sal-T response in elite male rugby players [9]. However, the same video footage failed to reproduce a significant hormonal change in healthy men [10], whereas a documentary film showing real-life aggression and violence did elevate Sal-T [7]. Thus, the situational context and emotional importance of the video footage to the assessed population may dictate whether a T response will occur. To our knowledge, no studies have examined the effect of a video clip showing aggressive sporting actions in male populations who differ in training level and background. Physical exercise also provides an effective stimulus for increasing blood (free, total, bioavailable) T and Sal-T levels in men, including continuous running [11, 12], interval running [12, 13], sub-maximal and maximal cycling [14], and weight training [15, 16]. Gonadal steroids are particularly responsive to sprint exercise [17–21], possibly due to the induction of important mechanical and physiological signals. Sprint exercise is also free of other constraints related to a video display (e.g. situational context, emotional value) and thus, could provide a more reliable stimulus for elevating T in both trained and untrained groups. However, no studies have compared the Sal-T responses to a psychological (video) and physical (sprint exercise) stimulus in men with different training backgrounds. This study compared the effects of a video clip with aggressive content and sprint exercise on the Sal-T responses in four groups of men: elite rugby players, recreational rugby players, mixed athletes involved in noncontact sports, and untrained controls. We hypothesized that sprint exercise would induce greater and more consistent Sal-T changes (increases) than the video clip, and that the video (due to the rugby-related aggressive content) would promote greater Sal-T responses in the elite rugby and recreational rugby groups than the mixed athletes and untrained men. Based on prior work [22, 23], we also examined the stability of the individual T responses across the different treatments.
Materials and methods Participants The study population (n = 53) comprised elite rugby players (n = 17, 21.5 ± 1.0 years, 186 ± 6 cm and 99.2 ±
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8.2 kg), recreational rugby players (n = 10, 21.1 ± 2.1 years, 183 ± 5 cm and 89.7 ± 8.7 kg), a mixed group of athletes involved in non-contact sports (n = 14, 23.1 ± 5.4 years, 184 ± 8 cm and 88.5 ± 8.9 kg) and untrained controls (n = 12, 21.0 ± 1.5 years, 180 ± 4 cm and 84.8 ± 7.2 kg). The elite rugby players were full-time professional athletes, whereas the recreational rugby players were involved in a local rugby competition. The mixed athletes were participating in various sports (cricket, football, bodybuilding and rowing) at either a local or national level, whilst the controls were not involved in any organized sport. Pre-screening assessments indicated that the study participants had no known disorders (e.g. endocrine, psychiatric) and were not taking any medicines or drugs (e.g. anabolic steroids) over the last 12 months that could influence the study outcomes. All data were collected with University ethics approval and participants signed informed consent. Experimental design Data from several experimental trials were collated for this study. In all trials undertaken, the participants were assessed using a crossover design after random allocation to three treatments: (1) watching a short video clip with aggressive content, (2) completing a short bout of sprint exercise and (3) a control session with no stimuli. Saliva samples were taken before and after each stimulus, along with time-matched samples across the control session. The samples were assayed for Sal-T levels and examined for changes within and differences between the study groups and treatments. Testing protocols Each treatment described above was separated by at least 3 days (up to a maximum of 7 days) and the participants were told to refrain from performing any exercise in the morning before each test. In addition, all intense exercise (especially for the lower body) was avoided in the 48-h period before testing to eliminate the confounding effects of fatigue. The study groups were assessed in the afternoon (between 1200 and 1600 h) to address expected time-of-day variation in T secretion and reactivity to physical or psychological stressors [24–26]. Furthermore, each participant completed their own assessments at a similar time of day (±1 h). Before the sprint assessment (see below), a familiarization session was performed to ensure that the participants were capable of completing the exercise procedures and to account for a pronounced stress response occurring from intense, unaccustomed exercise.
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Sprint exercise testing
Control testing
The participants arrived at the laboratory and sat quietly for 10 min in a stress-free environment. The exercise sprints were performed on a cycle ergometer (Monark 824E, Sweden) after a short warm-up with a light load. Three intermittent cycling protocols were used (5 9 10 s, 4 9 10 s, 3 9 10 s) with resistance loads ranging from 5 to 7.5 % of individual body mass. Short recovery periods (e.g. 40–60 s) were provided between each sprint repetition. The entire protocol was completed in 3–5 min. The participants were led through the protocols by at least two researchers, who also timed each event, modified the test and recovery loads, and provided feedback on when to start and stop each sprint. The testing protocols and equipment ensured that the same muscle groups were activated over a similar time period, with a mean exercising duration of 40 s. Similar sprint cycling protocols have been shown to raise blood T or Sal-T levels in men [17, 18, 20, 27]. Strong verbal encouragement was provided to ensure that the sprints were performed with maximal effort. A short cool down was performed after the final sprint consisting of slow pedalling without load. The participants then sat quietly in the laboratory until sampling.
Control data were taken from participants after an initial 10-min resting period. Time-matched saliva samples (as per the sprint and video treatments) were taken under similar conditions. This involved either watching a blank video screen or simply sitting quietly in the testing facility for the allotted time period. The participants remained seated throughout to prevent any residual effects of physical exercise (e.g. standing up, walking around) on the hormonal milieu and they were tested as individuals or in small groups. Again, there were no interactions between the study investigators and participants across the control session except to provide verbal instructions prior to testing and sample collection.
Video clip testing The participants sat quietly for 10 min in the laboratory before watching a short (4–5 min) video showing aggressive content of big rugby tackles and related aggressive play [9, 10]. This type of video footage was previously shown to acutely elevate male Sal-T levels [9]. Three video clips were used across the various trials, although the aggressive rugby content remained the same. The clips were taken from an online resource and played for the allotted timeframe (http://www.youtube.com/watch?v= i0dH5-2Yqmw, http://www.youtube.com/watch?v=UAaxe TowTd0 and https://www.youtube.com/watch?v=UTg1Xm 9CJgo). During the video presentation, the participants either sat by themselves to watch the video on a computer screen or they sat in small groups (up to 4) to watch the video on a larger projector screen. Group testing was unavoidable due to time constraints and facility availability. There was no communication between the investigators and subjects during the video presentations, or in the short period after, to eliminate the effects of social interactions on circulating Sal-T levels [28]. The only interactions occurred when simple verbal instructions were provided before the video presentation. At the end of the video, the participants sat quietly in the laboratory before subsequent sampling.
Salivary hormone testing Saliva samples were collected before each stimulus (after the rest period) and 15 min after to coincide with expected T changes in this media [5, 9, 27, 29], which are typically measured at 10–15 min post stimulus. The timing between the pre and post saliva samples was approximately 20 min. Saliva (1–2 ml) was collected by passive drool (not using Salivettes) and without stimulation (e.g. citric acid) into sterile containers and stored at -80 °C until assay. To account for dietary factors, each participant was instructed to maintain the same food and fluid intake before each session, and to refrain from eating or drinking 2 h before sampling [30]. Although the hydration state of individuals may have varied due to the self-selection procedures, hydration state does not influence blood T before, during or after exercise [31]. After thawing and centrifugation, the samples were analysed for T using a commercial immunoassay (Salimetrics LLC, USA). The detection limit for each plate was 6.1 pg/mL with inter-assay CV’s of 5–12 %, based on low and high control samples. Intraassay CVs on duplicate samples are typically less than 4 %. Samples for each participant were tested in the same assay to eliminate inter-assay variance. Statistical analyses The Sal-T measures were log transformed before analysis to normalize data distribution and reduce non-uniformity bias. The raw data are presented to allow comparisons with other studies. To assess the Sal-T responses, the delta change scores were calculated across each treatment (pre to post change presented as a mean percent value) and compared with paired T tests. The change scores in Sal-T were examined using a two-way (group 9 treatment) analysis of variance with repeated measures on the treatment variable.
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J Endocrinol Invest Table 1 Salivary testosterone levels pre and post the video, sprint and control treatments in the different male groups (mean ± SD) Video
Elite rugby players
Sprint
Control
Pre
Post
Pre
Post
Pre
Post 146
M
141
151
124
145
146
SD
22
27
16
18
27
24
Recreational rugby players
M SD
139 41
130 44
147 35
164 39
138 31
142 31
Mixed athletes
M
125
129
115
150
128
124
Untrained controls
SD
23
27
19
49
38
29
M
139
161
150
192
154
150
SD
37
63
53
81
67
60
The unit of measurement is pg/mL
Where appropriate, post hoc testing was conducted using the Tukey–Kramer test for unbalanced groups. The stability of the individual Sal-T responses to each treatment was assessed using Pearson correlations. Significance was set at an alpha level of p B 0.05.
Results Group demographics We found no significant differences in the mean age (F(3,52) = 1.403, p = 0.252) or height (F(3,52) = 1.954, p = 0.133) of the four male groups, but they differed in body mass (F(3,52) = 8.259, p \ 0.001). The elite rugby players were 9.5, 10.7 and 14.3 kg heavier than the recreational rugby players, mixed athletes and untrained controls (p \ 0.01), respectively. Testosterone responses Table 1 shows the descriptive Sal-T results for each group across the different treatments. In terms of the Sal-T responses (Fig. 1), the elite group showed a positive change across the video (6.9 ± 6.4 %) and sprint (17.1 ± 11.1 %) treatments (p \ 0.001). Conversely, SalT decreased after the video (-7.5 ± 11.0 %, p = 0.043) and increased with exercise (11.9 ± 15.9 %, p = 0.039) in the non-elite rugby group. The mixed athletes also had a positive Sal-T response to the sprints (27.6 ± 32.0 %, p = 0.006). In the control group, both the video (11.9 ± 13.5 %) and sprint (25.3 ± 23.6 %) stimuli produced an elevated Sal-T response (p \ 0.012). No changes in Sal-T levels were seen across the control sessions in any group (p [ 0.415). Examination of the Sal-T changes revealed a significant treatment effect (F(2,98) = 27.268, p \ 0.001) and
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Fig. 1 Percent change in salivary testosterone (Sal-T) levels across the video, sprint and control treatments in the different male groups (mean ± SD). Significant change from baseline #p \ 0.05, * p \ 0.01. 1 Significant from all of the control sessions, 2 significant from the elite rugby, recreational rugby and mixed athlete group video sessions, 3 significant from the elite rugby, mixed athlete and control group control sessions, 4 significant from the recreational rugby group video session p \ 0.05
group 9 treatment interaction (F(6,98) = 2.277, p = 0.042). The Sal-T responses to the sprint sessions (elite rugby, mixed athlete and control groups) were significantly greater than most of the video and/or control results (p \ 0.05), with the sprint and video outcomes also differing in the recreational rugby group (p \ 0.05). Following the video clip, the control group had a greater Sal-T response than the recreational rugby players (p \ 0.05). The individual pre and post Sal-T data for each treatment are plotted in Fig. 2. Testosterone interrelationships In the recreational rugby group, the individual Sal-T changes across the sprint and control treatments were strongly and significantly related (Table 2). The Sal-T responses to the sprint and video treatments were also correlated in the mixed athlete and control groups.
Discussion The physical stimulus employed in this study produced reliable changes (increases) in Sal-T levels across all of the male groups tested, thereby demonstrating a consistent population effect. In some cases the psychological stimulus also promoted elevated Sal-T levels, but the observed changes were generally smaller than the physical stimulus and the Sal-T responses were group dependent, not reflected at the population level. As hypothesized, sprint exercise provided an effective stimulus for increasing Sal-T levels and the mean changes in the elite rugby (17.1 %), recreational rugby (11.9 %),
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Fig. 2 Individual salivary testosterone (Sal-T) levels pre and post the video, sprint and control treatments in the elite rugby (a), recreational rugby (b), mixed athlete (c) and control groups (d)
Table 2 Correlations between the changes in salivary testosterone levels across the video, sprint and control treatments in the different male groups Sprint vs. video
Sprint vs. control
Video vs. control
Elite rugby players
r = 0.19, p = 0.475
r = 0.09, p = 0.732
r = -0.05, p = 0.851
Recreational rugby players
r = -0.48, p = 0.165
r = 0.82, p = 0.004
r = -0.54, p = 0.120
Mixed athletes
r = 0.72, p = 0.004
r = 0.25, p = 0.383
r = 0.02, p = 0.955
Untrained controls
r = 0.69, p = 0.013
r = -0.08, p = 0.810
r = -0.32, p = 0.303
mixed athlete (27.6 %) and untrained control (25.3 %) groups were similar irrespective of sporting involvement, or lack thereof. Our results also confirm the general finding that high-intensity sprint exercise can rapidly increase blood T (free, total) levels, along with more active metabolites (e.g. dehydrotestosterone), and Sal-T levels in male populations with different training backgrounds [17– 21, 27, 29]. Whilst physical conditioning and repeated
exposure to training stress (habituation) may reduce the acute T response to exercise [15, 32], the sprint cycling test is based on a voluntary maximal effort and as such may normalize for these factors. This could explain similar SalT responses in all groups tested despite expected differences in exercise capacity and tolerance, cycling workloads, and motivational drive. In response to the video, Sal-T was elevated in the elite rugby (6.9 %) and control groups (11.9 %), and the magnitude of change was similar to previous work [9, 10]. It was surprising that the untrained men were closest to the professional rugby players, whilst the recreational rugby players showed a negative video response (-7.5 %). We expected some specificity within a sport due to the video content; however, professional rugby differs considerably in training and competition from recreational sport so our observations could reflect perceptual and emotional factors relating to playing status, as well as content affiliation with only elite rugby actions shown. Reports of declining Sal-T levels in men after viewing a sad [9] and stressful video [5] further suggest that emotive content and individual perception are potential factors regulating the T response to a visual presentation.
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Different video clips have been shown to modify Sal-T (up to 10 %) in a small but defined group of professional rugby players [9]. As we speculated, this effect was not reproducible across a broader group of athletes in this work. This may again reflect the emotional value that an athlete group ascribes to the video shown, for example, watching a video of prior wins elevated Sal-T (42–44 %) but viewing a loss had no effect [8]. Watching erotic or sexual films can promote similar changes (15–35 %) in male T levels [5, 6, 33], which could be linked to emotional bonding and sexual arousal. A rise in Sal-T after watching a violent film probably reflects a general increase in physiological arousal [7]. Extending prior work showing a link between these Sal-T changes and muscle performance [9], future research could address whether similar T surges (induced by different stimuli) can differ in their functionality, or whether different stimuli can be combined to augment this response. It appears that sprint exercise (vs. video clip) provides a stronger and more reliable stimulus for increasing T availability. Other research comparing an exercise and cognitive stressor in children support our findings [34]. Given that the psychological stress response is determined by complex cognitive-appraisal mechanisms, it is not surprising that the blood or salivary responses of T can vary with the presentation of this type of stimuli [26, 34–38]. This could be partly explained by individual variation in hormonal reactivity [26], as we observed in the group results (see Fig. 2). One study did report small but similar increases in Sal-T in men completing a sprint exercise trial and when playing a violent video game [27]. However, a video game presents a very different psychological stimulus with a level of immersion that is also highly competitive in nature (i.e. outcome dependant), rather than simply sitting and passively watching a video clip. Any measured change in T could be explained by mechanisms involving secretion and/or synthesis rates, systemic changes (e.g. plasma volume, blood flow and clearance rates, binding protein levels and dissociation), and depending on the collection media, tissue activity (e.g. steroid metabolism, conversion) [1, 12, 14, 29]. The T response to physical exercise can be attributed to mechanical (e.g. cadence), metabolic (e.g. lactate) and/or autonomic (e.g. adrenaline) factors [18, 19]. Psychological factors contributing to T secretion include stress perception [35, 37], verbal actions (e.g. talking) [37] and any verbal feedback received [39]. The T changes occurring with either physical or psychological stimulation are also linked to other stress outcomes (e.g. cortisol, adrenocorticotropic hormone) [14, 38, 40] and hormonal predisposition (e.g. pre-test T levels) [26]. The relationships between the Sal-T responses to sprint exercise and the other treatments are novel findings, which
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also appear to be group dependent. Similar results were seen in other athlete groups across training sessions [23], competitions [22], as well as practice sessions and competition [41]. Collectively, these data confirm that T reactivity is somewhat stable on an individual level, especially when anchored against one or more physical stressors. By default, these results also confirm consistent variation in the blood T or Sal-T responses of individual athletes when exposed to the same exercise stressors [16, 42], and again evident in Fig. 2, which could indicate a genetic ability to respond to, and cope with, a stress challenge [4]. Indeed, prescribing workouts based on individual T variation have been used successfully to maximize short-term training adaptations [43]. Overall, our results suggest that sprint exercise provides a more reliable stimulus for modifying (increasing) Sal-T availability across broad populations and that these measurements are themselves stable. Therefore, this form of exercise could be used to assess T rhythmicity and dynamics [18, 27], to profile individual stress adaptations [42, 43], and modify physical performance [20]. The video shown produced less stable results that were group specific and probably dependent on a large number of factors such as emotive meaning, training status and specificity of sport, as well as cultural factors with respect to the video content. This makes it less suitable as a model for general population studies, but still interesting for examining the emotive content of a video as a modifier of T, behaviour and/or muscle performance [5, 8, 9]. We do acknowledge some of the study limitations, such as slight differences in the exercising protocols and the unbalanced groups. Although the general content of the videos was consistent across the study groups, the actual footage did show different rugby teams and players, along with different aggressive actions that could also influence how individuals perceive and subsequently respond to this. Furthermore, we did not address subject habituation to the video footage shown, nor did we monitor participant exposure to other psychological stressors before testing. The lack of data on other biomarkers of stress (e.g. cortisol) and the gonadal axis (e.g. gonadotropin-releasing hormone, luteinizing hormone) is other consideration. Research that addresses these issues would add value to the fields of behavioural endocrinology, sport and exercise, and general stress physiology. In conclusion, a physical stimulus involving sprint exercise gave a more reliable population response for Sal-T (increasing) in trained and untrained men than a psychological stimulus involving a video clip with aggressive content (increasing, decreasing and no change). Individual Sal-T reactivity was correlated across different treatments to indicate a level of stability in these measurements.
J Endocrinol Invest Acknowledgments We thank the participants, researchers and trainers who contributed to this study. This project was supported by the Elite Sport Performance Research in Training with Pervasive Sensing Programme [EP/H009744/1], funded by the Engineering and Physical Sciences Research Council, and the UK Sports Council.
16.
17. Conflict of interest
The authors declared no conflict of interests. 18.
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