Repeated exposure to water treadmill (WT) exercise could elicit kinematic responses reflecting adaptation to WT exercise. The study\'s aim was to compare the responses of a group of sport horses to a standardised WT exercise test (WTSET) carried out at three time points, week 0 (n = 48), week 20 (n = 38), and week 40 (n = 29), throughout a normal training programme incorporating WT exercise. Horses were recruited from the existing client populations of two commercial water treadmill venues for the purpose of this longitudinal, observational study. Limb, back, poll, wither, and pelvic kinematics were measured during the WTSET using videography, optical motion capture, and inertial motion sensors. Forelimb and hindlimb protraction increased (p < 0.001 for both), and forelimb and hindlimb retraction decreased (p < 0.001 for both) at week 40 compared to week 0. Caudal thoracic flexion-extension and lateral bend ranges of movement were greater at week 40 compared to week 0 (p < 0.001 and p = 0.009, respectively). Increased training speed was associated with increased craniocaudal poll movement (p = 0.021), decreased forelimb protraction (p = 0.008), and increased forelimb retraction (p = 0.021). In addition to characteristic changes in kinematics due to increasing water depth, regular WT exercise resulted in kinematic adaptation to movement in water. Factors such as the frequency of WT sessions and the type of session used with respect to depth and speed were seen to influence the nature of the adaptation. The results suggest that WT exercise sessions could be designed in accordance with specific training goals when used within a normal sport horse training programme.