OBJECTIVE To quantify fatigue-induced electromyographic changes in hind limb muscles in horses. ANIMALS 8 Thoroughbreds. PROCEDURES The left and right hind limb longissimus dorsi, tensor fasciae latae, gluteus medius, and biceps femoris muscles were instrumented for surface electromyography. Hoof strain gauges were attached to confirm stride cycle. Each horse was galloped on a treadmill (grade, 3%) at a constant speed (12.6 to 14.7 m/s) to achieve fatigue after approximately 360 seconds. Before and after this exercise, the horses were trotted at 3.5 m/s. At 30-second intervals during galloping an integrated electromyography (iEMG) value for a stride and the median frequency of muscle discharge (MF) in each limb were measured. The mean of stride frequency (SF), iEMG value, and MF of 5 consecutive strides at the start and end of galloping for the lead and trailing limbs were compared. For trotting, these variables were compared at 60 seconds before and after galloping. RESULTS The mean ± SD value for SF decreased over time (2.14 ± 0.06 to 2.05 ± 0.07 stride/s). In both the lead and trailing limbs, fatigue decreased the iEMG values of the gluteus medius and biceps femoris muscles but not those of the longissimus dorsi and tensor fasciae latae muscles. The MF did not change for any muscle during galloping with fatigue. The SF, iEMG value, and MF did not change during trotting with fatigue. CONCLUSIONS AND CLINICAL RELEVANCE Fatigue induced by high-speed galloping decreased the gluteus medius and biceps femoris muscles' iEMG values in Thoroughbreds. Fatigue of these less fatigue-resistant hind limb muscles would affect a horse's speed.