Consumption of β-hydroxy β-methylbutyrate (HMB) alters muscle composition and metabolism leading to strength and agility improvements in human athletes. To determine if HMB affects athletic performance and muscle function in horses, Thoroughbred geldings were fed a control (CON; n=5) or HMB (n=6) supplement for 6 wks prior to completing a standardized exercise test (SET). Gluteus Medius (GM) muscle biopsies were obtained before the SET for fiber typing. Heart rate (HR), biceps femoris (BF) and semitendinosus (ST) surface electromyograms (EMG) and fore and hind limb metacarpophalangeal joint angles were captured at the gallop of the SET. Results demonstrate that HMB supplementation increased (P < 0.05) the percentage of type IIA and IIA/X muscle fibers in the GM with a corresponding decrease (P < 0.05) in type IIX fibers. The percentage of type I fibers was unaffected by diet. Supplementation with HMB did not result in any measurable effects on performance or biomechanical properties by comparison to CON. Supplementation with HMB resulted in an increase (P < 0.05) in ST median frequency at speeds of 10 m/s and greater. Increasing treadmill speed resulted in an increase (P < 0.05) in stride length and the maximal proximal forelimb fetlock angle, and a decrease (P < 0.05) in stance phase time of the gait cycle. Integrated EMG increased (P < 0.05) with increasing treadmill speeds for both the BF and ST with the BF exhibiting greater (P < 0.05) iEMG values than the ST. In summary, HMB increased the percentage of type IIA GM fibers which did not translate into improved performance.