We examined the effect of treadmill speed and incline on O2 uptake (VO2), CO2 production, heart rate (HR), plasma lactate concentration, economy of locomotion, stride frequency, and stride length. A further aim was to examine the relationships between HR and VO2 and lactate and VO2 and whether these relationships vary with alterations in treadmill incline. The experiment was a latin square design, using five horses and five treadmill inclines (0, 2.5, 5.0, 7.5, and 10.0%). Fit Thoroughbred horses exercised for 4 min at 3 m/s at 0% slope, after which the treadmill was set to the allocated incline. Speeds tested ranged from 1 to 13 m/s. The relationships of VO2 and CO2 production with speed were curvilinear at 0 and 2.5% and linear at 5, 7.5, and 10% inclines. There was a linear relationship of HR and speed with a significant effect of incline. The plasma lactate concentration increased exponentially with speed, and there was a significant effect of incline. Stride length increased linearly and stride frequency increased in a curvilinear manner with speed but there was no effect of incline. There were linear relationships of HR with VO2 and HR with VO2 when expressed as percentage of maximum VO2 and maximum HR that were not affected by incline. The O2 cost of exercise on a 10% incline was approximately 2.5 times that for exercise on the flat. The strong relationship between the percentages of maximum HR and maximum VO2 indicates that over a wide range of exercise intensities the relative VO2 can be accurately predicted from measurements of HR.