Endurance ride competitions are long distance races of 40 to 160 km against the clock in phases that consist of a minimum of 16 to a maximum of 40 km, followed by a mandatory rest period, at least equal in minutes to the distance in km of the competition . A pre-inspection (PI) before the event and a veterinary inspection after each phase are compulsorily, and performed by a veterinary commission on every competing horse in an assigned area called the vet gate (VG). Upon examination of the heart rate recovery, metabolic status and regularity of gait, it is the veterinary commission that ultimately decides if a competing horse proceeds to the next phase and, after completion of the last phase, if the competitor merits the qualification and the finish line classification . Despite having the highest elimination rates of all equestrian disciplines and the introduction of stricter Fédération Equestre Internationale (FEI) rules, the recurrence of catastrophic injuries in endurance, particularly musculoskeletal [3–5], frustrates not only competitors, but also veterinarians. Moreover, the ongoing social license debate centred on the health and growing welfare concerns with equine athletes arising from the public and society [6, 7], largely reflected on social media , are jeopardizing not only horseracing, but equestrian sport in general, and endurance in particular.
As a result, there is a current quest for solutions to objectively quantify stress in horses during exercise. Biomarkers can be defined as a characteristic, substance or process which can objectively be measured and evaluated as an indicator of normal biologic and/or pathogenic processes and as a predictor of the outcome . Their usefulness in sports consists of providing accurate measurement about the response of an athlete to exercise undertaken. This is particularly important in equine athletes, because they cannot vocalize distress or pain as humans and cannot take decisions for themselves . However, biomarker testing poses some challenges in exercise physiology, i.e. limited sensitivity and specificity of single biomarkers to detect injury risk, interindividual variance in absolute values and relative changes. Results/ reliability are also dependent upon the context, such as previous training level and experience and type of exercise, for example an agility type exercise such as dressage versus and an effort type of exercise like racing. This can result in poorly defined reference ranges for athletes . Exercise is naturally a stressor and, as such, induces a biologic response to exercise that can be either an enhancer or a limiting factor for the sporting ability of an athlete and, therefore, determine the performance obtained . During competition, horses face a mixture of stressors including transportation , veterinary examinations , rider’s ability  a new and a noisy environment , separation from stable mates  and, specifically in endurance, exposure to large conglomerations of unfamiliar horses in large starts, and musculoskeletal pain from an injury that might arise . This complicates the interpretation of the levels of some stress biomarkers because it is hard to separate the impact of the different stressors on the welfare and performance of horses.
Cortisol has been studied exhaustedly in horses to determine stress levels and the response to different types, intensities and durations of exercise in sport and racehorses, including endurance. See the reviews from Hyyppä  and König v. Borstel et al.  for further information. Cortisol is the end result of the activation of the hypothalamic-pituitary-adrenal (HPA) axis as a response to any psychological or physical stressor. This response is influenced by intrinsic factors (age, gender, breed, inherited temperament, experience) and environmental extrinsic factors (competition setting, noise, type of imposed exercise, weather) . The first cortisol studies were performed using plasma, but the identification of free circulating, i.e. the truly biologically active component of blood cortisol in saliva, and its validation in horses by Peeters et al. (2011), made the collection of this biologic fluid, specially due to its non-invasiveness, much more popular. A circadian rhythm was demonstrated for salivary cortisol although a correlation with blood cortisol seems not to be proportional, most likely due to shifts between the free active component, the sole present in saliva, and the inactive bound component . Cortisol has been studied mostly in controlled environments, but also during endurance [22–24], show jumping [16, 25, 26] and dressage competitions [14, 27, 28]. In all studies effort induced the activation of HPA activity. Cortisol in saliva shows greater variations than in plasma [29, 30]. The highest increases from pre-exercise levels were registered in endurance (up to 1000%)  followed by eventing (240%) , showjumping (150–340%) and dressage (200%) competitions.
The changes in circulation associated with the HPA axis activation induce periorbital warming that can be quantified by thermal imaging cameras . The use of hairless vascularised areas such as the lacrimal caruncle to measure temperature minimises interferences of skin and coat colour, and environmental conditions . The rise in ocular temperature measured by infrared thermography (IRTOT) has been reported as a reliable indicator of short-term stress in animals and is often studied together with salivary cortisol measurements . IRT has identified the levels of stress induced by certain equestrian practices such as neck hyperflexion  or a tight noseband . More recently, IRTOT has been also studied in showjumping [35, 36] and dressage competitions , in Standardbred harness races  and in flat race Arabian and Thoroughbred horses in training . It has been generally accepted that the rise in ocular temperature represents an emotional response to stressors, including exercise , as opposed to a physiological response to physical demand of exercise, as proposed recently . IRTOT may represent a measure of emotive reactivity to effort, that can have a beneficial or detrimental effect on performance [15, 38]. For this reason it has recently been proposed as a selection tool to help identify emotional reactivity as a desirable, or undesirable, trait to performance according to the intended use of the horse [37, 38]. The complimentary use of salivary cortisol and IRTOT as non-invasive biomarkers of stress during endurance competitions could help characterise distress and physiological response to effort of endurance horses to endurance exercise in competition.
To our knowledge IRTOT alone or concomitantly with SC has not been studied before during endurance rides. This study aimed to determine trends in salivary cortisol (SC) and ocular temperature measured by infrared thermography (IRTOT), and its variation before and during endurance competitions in relation to the outcome and performance of competing horses.