OBJECTIVE: To evaluate alterations in skeletal muscle carnitine metabolism during exercise and training by measuring changes in plasma acylcarnitine concentrations in Standardbreds. ANIMALS: 10 Standardbred geldings with a mean +/- SD age of 20 +/- 2 months and weight of 384 +/- 42 kg. PROCEDURES: In a 32-week longitudinal study, training on a treadmill was divided into 4 phases as follows: phase 1, acclimatization for 4 weeks; phase 2, 18 weeks with alternating endurance and high-intensity exercise training; phase 3, increased training volume and intensity for another 6 weeks; and phase 4, deconditioning for 4 weeks. In phase 3, horses were randomly assigned to 2 groups as follows: control horses (which continued training at the same level as in phase 2) and high-intensity exercise trained horses. At the end of each phase, a standardized exercise test (SET) was performed. Plasma acylcarnitine, fatty acids, and lactic acid and serum beta-hydroxybutyric acid (BHBA) concentrations were assessed before and at different time points after each SET. RESULTS: Plasma lactic acid, total nonesterified fatty acids, 3-hydroxyisobutyric acid, and acetylcarnitine (C2-carnitine) concentrations significantly increased during SETs, whereas serum BHBA, plasma propionylcarnitine (C3-carnitine), and plasma butyryl- and isobutyrylcarnitine (C4-carnitine) concentrations decreased significantly, compared with those before SETs. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings indicated that the plasma acylcarnitine profile in horses likely reflects skeletal muscle carnitine metabolism following exercise, thereby providing a possible practical method to investigate potential disorders in carnitine metabolism in horses with myopathy.