The study aimed to investigate the effect of speed and incline on EMG activity in the brachiocephalicus muscle and the long and lateral heads of the triceps brachii muscle. Six horses were exercised on a treadmill at walk (1.7 m s-1), trot (4.0 m s-1) and right lead canter (7.2 m s-1) on a 0 and 8% incline. Kinematics (120 Hz) and electromyography (EMG) (2000 Hz) data were collected simultaneously from the left forelimb of each horse. Significant differences in relation to velocity and incline were identified using two-way ANOVA and post hoc Student–Newman–Keuls tests (P≪0.05). The degree of association between timing of peak EMG intensity and the timing of maximum protraction/retraction angles was assessed using ANCOVA. Increases in velocity led to an increase in stride length and reduction in stride duration. Exercise on the incline increased stance duration and decreased swing duration, while limb protraction/retraction increased. The time of peak EMG activity in the brachiocephalicus was highly related to time of maximum limb retraction (r2=0.84). The time of peak EMG activity in the long head of the triceps brachii was highly associated with time of maximum limb protraction (r2=0.87). Increases in velocity and incline both caused an increase in the intensity of the EMG signal from each muscle. Duration of EMG activity was prolonged in the long head of the triceps brachii muscle and in the brachiocephalicus muscle as velocity increased. Treadmill speed and slope therefore both alter the workload placed on forelimb muscles.