An Integrated Multi-Biomarker Perspective on Acute Exercise: Differential Orexin, Alpha-Amylase, and Cortisol Responses Across Modalities

Purpose: Acute exercise elicits coordinated neuroendocrine and autonomic responses that are strongly influenced by exercise modality and intensity. While cortisol and salivary alpha-amylase are widely used as peripheral indicators of hypothalamic–pituitary–adrenal (HPA) axis and sympathetic nervous system activation, respectively, salivary orexin remains an exploratory biomarker whose relationship with central orexinergic activity is not yet fully established, particularly in response to different exercise conditions. This study aimed to compare the acute salivary responses of orexin, alpha-amylase, and cortisol following distinct exercise modalities characterized by different physiological loads. Methods: A repeated-measures design was employed in a sample of physically active young women. Each participant completed four experimental sessions: high-intensity interval training (HIIT), combined exercise (CE), moderate-intensity continuous training (MICT), and yoga. Saliva samples were collected at baseline (T0), immediately post-exercise (T1), and during early recovery (T2 and T3). Biomarker concentrations were analyzed to assess temporal dynamics and modality-dependent differences. Results: All biomarkers showed modality-dependent responses. HIIT elicited the most pronounced increases in alpha-amylase and cortisol, reflecting robust sympathetic and HPA axis activation, whereas CE and MICT induced more moderate responses. Yoga was associated with minimal changes across all biomarkers. Orexin displayed a graded response across the exercise modalities, with higher post-exercise levels observed following HIIT compared to lower-intensity conditions. Temporal analyses revealed distinct kinetic profiles, with alpha-amylase peaking rapidly in the early post-exercise phase, whereas cortisol exhibited a more delayed response. Conclusions: These findings demonstrate that acute salivary responses of orexin, alpha-amylase, and cortisol are strongly modulated by exercise modality, supporting the importance of an integrated, multi-biomarker approach to characterize the physiological response to exercise. The inclusion of salivary orexin provides exploratory information on a potentially relevant arousal- and energy-regulation-related pathway, complementing established peripheral markers of autonomic and endocrine activation. This integrated perspective may contribute to a more comprehensive understanding of exercise-induced stress responses in young women.