The aim of this study was to investigate the effects of reduced muscle glycogen concentration on some physiological and metabolic responses during moderate intensity treadmill exercise in horses. Six Thoroughbred geldings were randomly allocated to 2 treatments (protocols A and B) or control in a 3 x 3 replicated Latin square design. In protocol A, horses performed low intensity exercise while horses in protocol B performed short bursts of high intensity exercise. Protocol A was designed to induce glycogen depletion mainly of slow twitch muscle fibers while protocol B aimed to deplete mainly fast twitch muscle fibers. Horses in the control group did not undergo exercise prior to the exercise test. Five hours after glycogen depletion, horses performed treadmill exercise at 60% VO2max at a treadmill slope of 10% until fatigue (20-30 min). The induced glycogen depletion prior to exercise had no significant effect on plasma glucose, insulin, or lactate concentrations during the exercise test, and there was no effect on glycogen utilization rate, although respiratory exchange ratios were lower in the glycogen-depleted groups. The VO2, heart rate and central blood temperature did not vary significantly between the protocols A and B and control throughout the exercise test. It was concluded that 20-30% depletion of glycogen concentration in the middle gluteal muscle resulted in a shift towards fat metabolism, but does not significantly affect heart rate, oxygen uptake, or concentrations of plasma glucose and lactate during moderate intensity exercise.