The current study aimed to investigate whether peripheral modulators of serotoninergic function and neurohumoral factors' changes in athletic horses during an official jumping competition, and to evaluate their relationship with the physical performance of competing horses. From 7 Italian Saddle mares (6-9 years; mean body weight 440 ± 15 kg), performing the same standardized warm-up and jumping course during an official class, heart rate (HR) was monitored throughout the competition. Rectal temperature (RT) measurement, blood lactate and glucose concentration, serum tryptophan, leucine, valine, the tryptophan/branched-chain amino-acids ratio (Try/BCAAs), dopamine, prolactin, and non-esterified fatty acids (NEFAs) were assessed before the exercise event (T0), at the end of the competition stage (5 min ± 10 s following the cessation of the exercise, T), and 30 min after the end of competition (T). Highest HR values were recorded during the course and at the outbound ( < 0.0001); blood lactate concentration and RT increased after exercise with respect to the rest condition ( < 0.0001). Lower leucine and valine levels ( < 0.01), and higher tryptophan, Try/BCAAs ratio, and NEFAs values were found at T and T with respect to T0 ( < 0.0001). A higher prolactin concentration was found at T and T compared to T0 ( < 0.0001), whereas dopamine showed decreased values after exercise compared to rest ( < 0.0001). Statistically significant correlations among the peripheral indices of serotoninergic function, neurohumoral factors, and athletic performance parameters were found throughout the monitoring period. The findings provide indirect evidence that the serotoninergic system may be involved in fatigue during jumper exercise under a stressful situation, such as competition, in which, in addition to physical effort, athletic horses exhibit more passive behavior.