Standardised exercise tests were performed at 2 different tracks and on an uninclined treadmill during the same week to determine the influence of exercise surface on different measured variables such as heart rate (HR), blood lactate concentration, packed cell volume, stride frequency, stride length, gait symmetry and regularity and on different derived physiological variables such as the speed at a HR of 200 beats/min (V200), the speed at a blood lactate concentration of 4 mmol/l (V4), the speed at a maximal HR (VHRmax). Five French Trotters, age 3 years, in training for 3 months prior to the test, performed 3 exercise tests on a training track (Test 1), a racetrack (Test 2) and an uninclined treadmill (Test 3). Test 1 utilised 3 steps each of 3 min at speeds of 490, 560 and 630 m/min. Tests 2 and 3 utilised the same speeds and a fourth step in which the horse was accelerated for 30 s to speed approaching maximal. No significant differences (P < 0.05) were found for the physiological and locomotor variables between the 2 tracks. In contrast, there was a significant difference (P < 0.05) for these variables between the tracks and the treadmill, horses showing lower heart rate and blood lactate response, reduced stride frequency and increased stride length and regularity on the uninclined treadmill. We concluded that this standardised exercise test was repeatable on various tracks even when the surface and geometry vary. In contrast, both physiological and locomotor variables were different when comparing the tracks with the uninclined treadmill.