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This study examines seasonal changes in muscle and heart parameters in eventing horses over the course of a competition season. Blood levels of the enzymes creatine kinase (CK) and aspartate aminotransferase (AST) as well as the heart muscle-specific concentration of cardiac troponin I (cTnI) were measured before (pre), 30 min (p30) and 24 h (p24) after competitions. Creatine kinase (CK: median pre-competition = 175 U/L, 30 min post = 221 U/L, 24 h post = 140 U/L), aspartate aminotransferase (AST: pre = 319 U/L, p30 = 335 U/L, p24 = 333 U/L), and cardiac troponin I concentration (cTnI: pre = 0.006 ng/mL, p30 = 0.011 ng/mL, p24 = 0.007 ng/mL) exhibited partial normalization by 24 h post-exercise but at the same time demonstrated significant seasonal variation (p < 0.001). Echocardiographic assessments revealed sustained high-level myocardial velocities, with occasional modest seasonal declines. A significant correlation was identified between cTnI levels and early diastolic myocardial velocity (Em) (Spearman's Rho: pre-exercise 0.323, Rho p30: 0.357), whereas a negative correlation was manifest at 24 h (Spearman's Rho = -0.300). These findings suggest a heightened sensitivity of diastolic myocardial velocity to myocardial injury. Given that diastolic dysfunction frequently constitutes an early manifestation of myocardial compromise, our results underscore the utility of biomarkers alongside myocardial velocity measures as valuable tools for the early detection of subclinical fatigue in high-performance sport horses.