Although it is known that equine-assisted interventions (EAI) have beneficial effects on human participants, the effects on horses have not been extensively studied. This study investigated the effects of EAI with military veterans with post-traumatic stress disorder (PTSD), on the heart rate (HR), heart rate variability (HRV), and behaviour of the involved horses. In total six different horses were deployed multiple times during a 12-week program of EAI for veterans with PTSD. The HR and HRV were measured during baseline conditions, free activities and directed activities.