Simple SummaryThe muscular adaptations that facilitate the differing biomechanical functions of the leading (Ld) and trailing (Tr) limbs during canter in horses remains largely unknown. We conducted the first comparative study of muscle activation and movement within the leading and trailing fore- (F) and hindlimbs (H) during overground canter. Surface electromyography and three-dimensional motion capture data were collected from the right fore- and hindlimbs, as well as the splenius muscle, of ten horses ridden in left- and right-lead canter, when the limbs functioned as TrF/TrH and LdF/LdH, respectively. The TrH is first to make ground contact and exhibited significantly greater gluteal activation than LdH to stabilize the more extended hip joint and to generate greater limb retraction and a strong forward push-off during stance. Then, during TrF and LdH diagonal support, bilateral splenius activation occurred, possibly to counteract downward head and neck movement. The LdF was the last to make contact and was more protracted than the TrF through greater elbow flexion during swing, but triceps activity did not significantly differ between forelimbs. Inter-limb differences in movement and muscle activity provide an objective justification for working the horse equally on both canter leads to promote balanced muscular development.AbstractThis study compared muscle activity and movement between the leading (Ld) and trailing (Tr) fore- (F) and hindlimbs (H) of horses cantering overground. Three-dimensional kinematic and surface electromyography (sEMG) data were collected from right triceps brachii, biceps femoris, middle gluteal, and splenius from 10 ridden horses during straight left- and right-lead canter. Statistical parametric mapping evaluated between-limb (LdF vs. TrF, LdH vs. TrH) differences in time- and amplitude-normalized sEMG and joint angle–time waveforms over the stride. Linear mixed models evaluated between-limb differences in discrete sEMG activation timings, average rectified values (ARV), and spatio-temporal kinematics. Significantly greater gluteal ARV and activity duration facilitated greater limb retraction, hip extension, and stifle flexion (p< 0.05) in the TrH during stance. Earlier splenius activation during the LdF movement cycle (p< 0.05), reflected bilateral activation during TrF/LdH diagonal stance, contributing to body pitching mechanisms in canter. Limb muscles were generally quiescent during swing, where significantly greater LdF/H protraction was observed through greater elbow and hip flexion (p< 0.05), respectively. Alterations in muscle activation facilitate different timing and movement cycles of the leading and trailing limbs, which justifies equal training on both canter leads to develop symmetry in muscular strength, enhance athletic performance, and mitigate overuse injury risks.