Four mature horses (3 Standardbreds and 1 Thoroughbred) were used in a 4 × 4 Latin square design experiment to investigate the effect of time of concentrate feeding prior to exercise on the metabolic response to exercise. Horses were fasted overnight and then given no feed (fasted) or 1 kg corn grain 1, 3 or 5 h before exercise. Exercise consisted of a warm-up (1600 m at 6.0 m/s) and walk (800 m) followed by a high intensity phase (400 m at increasing speed and 1600 m at 11 m/s; 2% grade). During exercise, plasma glucose decreased in horses fed 1 or 3 h before exercise (P<0.05), but increased in horses fasted before exercise (P<0.05). Plasma insulin declined (P<0.05) during exercise in horses fed 1 or 3 h before exercise but did not change in horses that were fasted or fed 5 h before exercise. For all treatments, peak plasma free fatty acids (FFA) occurred during the walk phase and FFA decreased (P<0.05) during the last 2000 m (11 m/s; 2% grade). At the onset of exercise and during much of the exercise test, plasma FFA were highest in horses that were fasted for 16 h before exercise. Plasma lactate increased during the exercise test but was not affected by treatment (P>0.05). These data indicate that time of feeding prior to exercise can affect metabolic responses of horses to exercise, which could ultimately alter performance. In addition, researchers should consider that time of feeding may affect the interpretation of data from experiments examining the effects of diet and/or training on exercising horses.