The aim of this study was to investigate the behavioural response of horses to rein contact and the movement of the riders' hands through analysis of data from horses ridden at two different head and neck positions. It was hypothesised that the riders' hand movements and rein tension would generate behavioural responses from horses and that these responses would be more marked when horses were ridden 'on the bit' than when unrestrained. Data were collected from seven dressage horse/rider combinations at sitting trot on a high speed treadmill. Kinematics were recorded using a 12-camera, infrared-based opto-electronic system. Three horses wore a rein tension meter. Behavioural registrations were made from video. Behavioural responses included lip movement, mouth movement, open mouth, change in ear position, head tilt and tail movement. Mouth movements were associated with the suspension phase of the trot. Head and neck position was non-significant in the final models, while rein tension and the distance between the rider's hand and the horse's mouth were related to mouth movements. Interactions between horses and riders are complex and highly variable.