Exercise-specific plasma proteomic signatures in racehorses:Candidates for training adaptation and peak load monitoring

Abstract

Background: Racehorses undergo profound physiological changes with training andcompetition, but current biomarkers inadequately capture the complex moleculardynamics of exercise. This study aimed to identify novel plasma biomarkers of train-ing adaptation and peak load using high-throughput proteomics.Objectives: We hypothesised that systematic training and racing induce distinctplasma proteomic signatures, enabling the discovery of candidate biomarkers linkedto training status, oxidative stress, inflammation and metabolic remodelling.Study Design: In vivo longitudinal study.Methods: Forty-nine Arabian and Thoroughbred racehorses underwent standardisedhigh-intensity training. Plasma samples were collected at rest, immediately post-exercise and after recovery during three phases: initial training (T1), mid-season con-ditioning (T2) and race-phase (R). In total, 314 samples were analysed using tandemmass tags based quantitative proteomics and Orbitrap mass spectrometry. Proteinabundance changes were assessed with multiple-testing correction (q < 0.05), andpathway enrichment was performed using STRING and ShinyGO.Results: Proteomic responses differed by phase. T1 showed broad activation of inflam-matory (S100A8/A9), antioxidant (superoxide dismutase 1, catalase) and metabolic pro-teins (glucose-6-phosphate dehydrogenase, phosphoglycerate kinase 1). T2 displayed amore refined profile with remodelling and redox regulators (decorin, thymosin β4, gluta-thione S-transferase). Racing elicited the strongest response, with over 100 up-regulatedproteins linked to energy metabolism, oxidative defense and cytoskeletal adaptation.Several proteins: including S100A8, thymosin β4, prothymosin-α, cofilin-1 and lipocalins,were consistently modulated across phases, highlighting their biomarker potential.Main Limitations: Breed imbalance and incomplete follow-up sampling may affectgeneralisability. Validation in larger, diverse cohorts with targeted assays is required.Conclusions: This study identifies a panel of promising plasma proteins as candidatebiomarkers of exercise adaptation and overload in racehorses. These findings maysupport improved monitoring of performance, training load and early detection ofovertraining in equine athletes.