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Monitoring exercise-induced stress is therefore essential to understand racehorses \u2018responses to effort and to ensure their welfare. Stress perceived by the horse during physical activity can be measured using various indicators, including stress-related hormones such as cortisol and adrenaline, and other neuromodulators such as serotonin, all involved in the stress response and its regulation. Another approach to assess physiological and emotional responses to stimuli such as exercise is through behaviours and facial movements. In this study, we aimed to 1) evaluate the changes in these three hormones following a trotting exercise, 2) determine the changes in behaviour and facial movements in response to the same exercise and 3) investigate potential relationships between hormonal variations and specific behavioural patterns that could serve as indicators of exercise-induced stress in horses. Fourteen French Standardbred horses from two stables were monitored over one day. In the morning, they performed an interval training trotting exercise. Behaviours and facial movements were recorded via video for 2\u2009min 30 both before and just after exercise. Saliva and blood samples were collected at four time points: before exercise, just after exercise, 1\u2009h post-exercise and 24\u2009h post-exercise to assess salivary cortisol, and serum concentrations concentration of adrenaline and serotonin. Results showed significant post-exercise increases in all three hormones with peak concentrations observed immediately after exercise, and elevated cortisol and adrenaline levels persisting one hour later. These variations are consistent with normal physiological responses to physical effort, reflecting activation of regulatory systems rather than necessarily indicating negative stress. However, inter-individual variability in the magnitude of these responses suggests that horses did not all experience the exercise in the same way, highlighting potential differences exercise-induced stress. In terms of behaviour, horses exhibited higher frequencies of facial movements, particularly mouth movements, after exercise than before. Moreover, increases in serotonin and adrenaline concentrations were positively associated with agitation-related behaviours (pawing and head turning) and mouth movements. Overall, our findings suggest that a behavioural profile characterised by increased agitation and mouth movements may reflect a post-exercise arousal response in French Standardbreds. These behaviours, in association with hormonal changes, could provide a useful non-invasive tool to assess to assess horses\u2019 response to exercise, and potentially exercise-induced stress. However, further studies are needed to confirm this interpretation, particularly by investigating the potential effects of post-exercise management practices such as cross-tying, which may induce frustration.<\/jats:p>","DOI":"10.1371\/journal.pone.0328430","type":"journal-article","created":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T18:27:15Z","timestamp":1762367235000},"page":"e0328430","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Exploring the association between stress-related hormonal changes, behaviours and facial movements after an interval training exercise in French 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