Water treadmill (WT) exercise is frequently used for training/rehabilitation of horses but the effect of WT exercise on short-term movement patterns is yet to be investigated. The objective of this study was to determine the immediate effect of WT exercise on overground limb and back kinematics. Six horses (mean ± standard deviation., age 15±6.5 years, height 164±2 cm and weight 539±37 kg) walked twice in a straight line, led from both sides, before and after a standardised WT exercise session (19 min duration; speed 1.6 m/s; water depths: 0.0/7.5/21.0/32.0/47.0 cm) on a flat concrete surface. Horses wore five inertial-measurement-units to determine poll/wither/pelvic displacement, and 10 anatomical markers to determine fetlock/carpal/tarsal joint angles at specific stride points. Degree of mediolateral tarsal oscillation during stance was graded. Wilcoxon-signed-rank tests were used to investigate differences between pre- and post-WT exercise for each variable. Post-WT exercise, there was a significant decrease in hindlimb fetlock extension at mid-stance compared with pre-WT exercise. No significant changes in movement patterns of the poll/withers/pelvis were detected post-WT exercise. In all horses there was greater mediolateral tarsal oscillation during the stance phase of the stride post-WT exercise, which could relate to muscle fatigue. The results suggest that a 19-min WT session has an effect on immediately-following overground in-hand walking locomotion patterns. Further work is required to determine the duration of this effect, and how different WT speeds and water depths affect locomotion patterns.