High intensity interval training (HIIT) has been recognized as an alternative to classic continuous endurance training, bringing about similar or even greater performance and health benefits (Gibala et al., 2006). It is characterized by short, repeated bouts of high intensity efforts, separated by recovery periods (Gibala et al., 2012). The main physiological changes produced by HIIT include improved substrate utilization (Perry et al., 2008), increased maximal oxygen uptake, improved cardiac and endothelial function (Little et al., 2011; Tjonna et al., 2013), and acute metabolic stress and hormonal responses (Wahl et al., 2013). However, it remains unresolved whether the training load of several HIIT sessions within a short period of time compromises the mucosal immune function.
Exercise causes a constant physiological and psychological stress in elite athletes. Ninety-five percent of infectious pathogens enter through the mucosa of the upper respiratory tract (Neville et al., 2008; Spence et al., 2007), ultimately reducing training effects and athletic performance (Pyne et al., 1998; Gleeson et al., 2001). One of the main players involved in immune system regulation is immunoglobulin A (IgA), being the first line of defense and an indicator of mucosal immune system (Neville et al., 2008). Previous studies have reported reductions in IgA levels following strenuous and repetitive exercise, which might be mediated by training volume and intensity (Trochimiak and Hubner-Wozniak, 2012). The decreased levels in this marker of immune function could lead to the so-called "open window", during which athletes are more susceptible to upper airway infections (Kakanis et al., 2010). Decreases in salivary IgA concentrations have been reported after both acute and chronic exercise as well as after strenuous and high-volume exercise (Nieman et al., 2002). For instance, IgA concentrations were reduced by 30% after three Wingate tests (MacKinnon and Jenkins, 1993) and by ~50% immediately post-marathon race (Nie-man et al., 2006). A 75% decrease in IgA has also been shown after a soccer match in elite male soccer players (Pe-nailillo et al., 2015).
Salivary alpha amylase (sAA) has been described as the most sensitive stress response marker due to exertion as it is directly produced in saliva (Papacosta and Nassis, 2011; Rohleder et al., 2009). Acute increases in sAA levels have been reported following strenuous activities such as short progressive tests to exhaustion (Allgrove et al., 2008; de Oliveira et al., 2010), and rowing (Kivlighan and Granger, 2006). The increase in sAA levels has been proposed to counteract the reductions in IgA levels, as well as the immune depression commonly observed following strenuous activities (Gatti and De Palo, 2011).
Exercise has been proposed to influence the regulation of testosterone and cortisol levels (Doan et al., 2007; Gatti and De Palo, 2011). Short bouts of endurance exercise tend to increase testosterone levels in both females (Consitt et al., 2002; Cumming et al., 1987; Nindl et al., 2001) and males (Hayes et al., 2015). Elevated circulating cortisol levels have been reported after intense intermittent or continuous endurance exercise in males (Kraemer et al., 1999; Sari-Sarraf et al., 2006) and females (Consitt et al., 2002). Gender differences in cortisol and testosterone levels have been reported in volleyball tournaments, which seem mainly associated to the intensity of the efforts during the matches (Penailillo et al., 2018).
Although the effects of diverse exercise types on the endocrine and immune responses have been reported, there is still a paucity of data on the responses after a HIIT session and whether these responses differ between males and females. Therefore, besides the well documented effects of HIIT training on health and sport performance, the prophylactic role of this type of training needs to be investigated, considering possible gender differences. The aim of this study was to assess the effect of an acute HIIT session on endocrine- and mucosal immune function of male and female endurance athletes. To this end, endocrine and immune responses were monitored through the changes in salivary testosterone, cortisol, IgA and sAA concentrations after a HIIT session. We hypothesized that one HIIT session would induce a decreased immune response and favour catabolic processes through a decrease in the Testos-terone/Cortisol ratio in the early recovery period in both males and females
Subjects' characteristics are presented in Table 1. Ten male and ten female long distance national level runners volunteered to participate. The inclusion criteria included: aged between 18 - 40 years, participating in [greater than or equal to] five training sessions/week, > 4 years of systematic long distance running training, national competition experience, peak oxygen uptake (V[O.sub.2peak]) [greater than or equal to] 50ml/kg/min for males and [greater than or equal to] 40ml/kg/min for females, and no recent musculoskeletal injuries (continuous training in the 6-month period before intervention), cardiovascular or infectious diseases. Female participants were evaluated in the follicular phase of their menstrual cycle to minimize potential effects of estrogen on muscle stress caused by exercise (Enns et al., 2007). The study conformed to the Code of Ethics of the World Medical Association (Declaration of Helsinki) and was approved by the University's Ethics committee.
Body composition and V[O.sub.2Peak] measurements
Body composition was assessed by body mass index and body fat percentage as estimated from the sum of six skin folds (Slimguide Caliper) (Legaz Arrese et al., 2006). [VO.sub.2max] was measured using an incremental running protocol on a treadmill (h/p/cosmos pulsar[R] 3p, Germany), during which gas exchange was assessed using a breath-bybreath gas analyser (Ergocard, Medisoft, Belgium). The incremental running protocol was started at 11km/h for male and 10km/h for female participants. The intensity was increased by 1km/h every 2min, with a fixed slope of 1.5%. [VO.sub.2max]criteria were: 1) Plateau in V[O.sub.2] despite increase in workload, 2) Respiratory exchange ratio (RER) [greater than or equal to] 1.10, and 3) Maximal heart rate expected for age (220 bpm - age). Tests were terminated when two of these three criteria were met (Castro-Sepulveda et al., 2019).
The participants attended the laboratory to perform an acute HIIT 5-7 days after the V[O.sub.2peak] test. They were instructed to refrain from any intense physical activity, dietary supplementation and pharmacological intake in the 48h prior to the intervention. Furthermore, they were not prohibited from consuming tobacco, alcohol and caffeine in the 12h prior to the HIIT session. A standardized diet with a high carbohydrate (125g) and protein content (21g) was provided for breakfast and lunch on the day of the intervention. The HIIT session was performed after two hours of fasting, between 3:00pm and 6:00pm, to minimize hormonal fluctuations. The session consisted of ten, 4-min running intervals at an intensity that elicited 90% of [VO.sub.2peak], which corresponded to a speed of 15.9 [+ or -] 6.8 km/h for male and 14.6 [+ or -] 4.7 km/h for female participants. Each interval was separated by two min of rest. The protocol included a 3-min warm-up and a 3-min cool-down at 45% of [VO.sub.2peak] at an average speed of 7.1 [+ or -] 0.4km/h for male and 7.8 [+ or -] 0.4 km/h for female participants. During the rest periods, the participants were instructed to remain on the side of the treadmill and be prepared to resume exercise on the treadmill 30s before the next interval. Participants were supervised by the same examiner, who monitored their heart rate (HR) to ensure that the exercise was performed at the prescribed intensity. The coefficient of variation (CV) for the present study was 5.3% for HR. The rate of perceived exertion (RPE) was recorded after completing each interval. Participants were asked to rate their perceived exertion on a Borg 6-20 scale (Moreira et al., 2009). The CV for the present study was 20.9% for RPE. Blood lactate...