Hippotherapy in patients with neuromuscular dysfunction creates high focal pressure on the pony's back due to bareback riding and an asymmetrical riding position. This study aimed to investigate the acute effect of asymmetrical bareback riding on the pony's spinal kinematics, blood lactate, serum creatine kinase, heart rate, and temperament score. Eight ponies were selected, and they were walked on a treadmill for 45 min on each experimental day, including warm-up (5 min), weight-loading by mannequin (30 min), and cool-down (10 min) sessions. During the weight-loading session, three different weight distributions on the pony's back were applied between the left and right side: 50:50 (treatment M), 70:30 (treatment L), and 30:70 (treatment R) on the first, second, and third day of the experiment, respectively. The spinal kinematics at the end of the weight-loading session revealed a slight reduction in range of motion in both flexion-extension and lateral bending during treatment R. Stride length and stride duration showed no differences between treatments. The levels of blood lactate and serum creatine kinase and results of a back examination were normal. Heart rates and temperament scores revealed that all ponies were calm throughout loading of the mannequin. This information suggests that asymmetrical bareback riding did not cause acute or serious back injury, which indicates good equine welfare in ponies used for hippotherapy.