The purpose of the present study was to determine the influence of fitness on cardiac function, particularly on left ventricular function parameters. Fifteen healthy ‘three-day event’ warmblood horses were examined at rest and immediately after high-speed treadmill exercise (3% incline, 3 min 1.8 m s−1, 3 min 4 m s−1, 3 min 5 m s−1, 3 min 6 and 3 min 7 m s−1, 1.5 min 8 m s−1). Horses were divided into two groups. Group 1 consisted of nine conditioned horses and group 2 included six unconditioned horses. Left ventricular dimensions and function were acquired using standardized echocardiographic indices. To assess the level of fitness, heart rate and blood lactate concentration were determined at rest and immediately after exercise. The group of conditioned horses showed a significantly lower blood lactate concentration (mean value 2.39 mmol l−1) after high-speed treadmill exercise than did the group of unconditioned horses (mean value 3.81 mmol l−1), which clearly revealed the difference in fitness between the two groups. During exercise the heart rate was not significantly different between both groups. Only in the recovery phase did the trained horses show a significant faster decrease in heart rate than did the untrained horses. Mean heart rate during echocardiography immediately after exercise (within the first 2 min) was 105 bpm in the group of trained horses and 113 bpm in the group of untrained horses.Within each group of horses, several echocardiographic parameters differed significantly between resting values and values after treadmill exercise. Particularly, in the group of trained horses, 17 out of 30 echocardiographic parameters (most diastolic) differed significantly between rest and exercise. In the group of untrained horses, only six out of 30 parameters were significantly different. At rest, left ventricular diameter at the apex cordis, left ventricular free wall at papillary muscle level, left ventricular volume and stroke volume, as well as fractional shortening (at the apex cordis and at papillary muscle level) were significantly different between both groups. After treadmill exercise comparison of echocardiographic parameters of the conditioned to those of the unconditioned animals showed no significant differences. In the present study, data have been provided for stress echocardiography in conditioned and unconditioned warmblood horses without any disorders of the cardiovascular system.