BACKGROUND: Prolonged exercise in human athletes is associated with transient impairment of left ventricular (LV) function, known as cardiac fatigue. Cardiac effects of prolonged exercise in horses remain unknown. OBJECTIVES: To investigate the effects of prolonged exercise on LV systolic and diastolic function in horses. ANIMALS: Twenty-six horses competing in 120-160 km endurance rides. METHODS: Cross-sectional field study. Echocardiography was performed before and after rides, and the following morning, and included two-dimensional echocardiography, anatomical M-mode, pulsed-wave tissue Doppler imaging, and two-dimensional speckle tracking. Correlation between echocardiographic variables and cardiac troponin I was evaluated. RESULTS: Early diastolic myocardial velocities decreased significantly in longitudinal (baseline: -17.4 ± 2.4cm/s; end of ride: -15.8 ± 3.2cm/s (P = .013); morning after: -15.4 ± 3.0cm/s (P = .0033)) and radial directions (-32.8 ± 3.4cm/s; -28.1 ± 5.8cm/s (P < .001); -26.4 ± 5.5cm/s (P < .001)). Early diastolic strain rates decreased significantly in longitudinal (1.58 ± 0.27s(-1) ; 1.45 ± 0.26s(-1) (P = .036); 1.41 ± 0.25s(-1) (P = .013)) and circumferential directions (2.43 ± 0.29s(-1) ; 1.96 ± 0.46s(-1) (P < .001); 2.11 ± 0.32s(-1) (P < .001)). Systolic variables showed ambiguous results. No correlations with serum cardiac troponin I concentrations were evident. CONCLUSIONS AND CLINICAL IMPORTANCE: Prolonged exercise in horses is associated with impaired LV diastolic function. Reduced ventricular filling persisted for 7-21 hours despite normalization of biochemical indicators of hydration status, indicating that the observed changes were not entirely related to altered preload conditions. The clinical relevance of cardiac fatigue in horses remains uncertain.