The hypothalamic-pituitary-adrenal (HPA) axis is a key neuroendocrine pathway responsible for the adaptive stress response triggered by physical exertion. Exercise acts as a major challenge to physiological balance, stimulating the concurrent secretion of ACTH and cortisol depending on the exercise features in sport horses. Considering this, the present study aimed to assess fluctuations in circulating adrenocorticotropic hormone and cortisol levels in Standardbred trotters following both training and race efforts, while accounting for age and sex differences. Specifically, it sought to quantify the extent of ACTH and cortisol elevation during maximal exertion in racing and to contrast hormonal responses under two exercise intensities: routine training and competitive performance. Ten healthy Standardbred horses (three mares and seven stallions) participated in two distinct conditions—non-competitive training and subsequent official racing. Among them, four were 2 years old and six were 3 years old. Both training and racing produced significant effects on ACTH (p < 0.01) and cortisol (p < 0.01) levels. Relative to training, racing caused markedly higher ACTH concentrations at rest (p < 0.001), and at 5 (p < 0.01) and 30 minutes (p < 0.001) post-exercise, while resting cortisol levels were lower (p < 0.01) following the race. After racing, both 2- and 3-year-olds displayed increased ACTH levels at 5 and 30 minutes (p < 0.01), and males exhibited elevated ACTH values at the same time points (p < 0.01). The differing contexts and intensities of the two exercise conditions appear to influence the HPA axis response directionally, reflecting both adaptive capacity to variable physical stress and preparatory adjustments to stimuli. In summary, training and racing elicited distinct HPA axis activations, wherein emotional arousal and physical maturity seemed to shape adaptive outcomes. Since ACTH and cortisol levels in mature horses exhibit wide variability, further research is necessary to clarify the factors modulating these endocrine patterns and their function as indicators of stress adaptation in equines.
