With the increase in the number of horses being used in Equine-Assisted Activities and Therapies (EAAT) programs and with the increasing concern for animal welfare, it is important to understand the impact of such interventions on the stress level and quality of life for the horses involved. The purpose of the present pilot study was to test the hypothesis that participation in EAAT would acutely alter physiological markers of stress and well-being, including plasma cortisol, plasma oxytocin, and heart rate variability (HRV), in horses and that symptoms of posttraumatic stress disorder (PTSD) would be reduced after five sessions of EAAT in veterans who had previously been diagnosed with PTSD. Nine healthy geldings, of various breeds, ages 10–23 years, conditioned and experienced as therapeutic riding horses, were selected to participate in the study. Of these, seven were selected at random to wear electrocardiogram units, and all nine were used for blood sampling to measure plasma cortisol and oxytocin. Each horse was randomly assigned to partner with a veteran for five EAAT sessions, 1 hour in duration. A standing control was conducted on a later date on which horses did not participate in EAAT. Measurement after 5 days of EAAT was conducted immediately after the end of the last session on day 5 using the Brief Symptom Inventory and the PCL-5 (a 20 item self-report measure of the Diagnostic and Statistical Manual of Mental Disorders - 5 for symptoms of PTSD). Two way repeated measure analysis of variance showed no significant day by time interactions for plasma cortisol (P = .821) or oxytocin (P = .861). There was a significant day by time interaction (P = .006) for heart rate (HR); where on day 1, HR (bpm) was significantly lower during the interaction with the veterans. There were no significant differences in HRV variables. Posttherapy measures in PTSD symptoms in veterans were significantly reduced except for interpersonal sensitivity (P = .08) and phobic anxiety (P = .17). There was an effect of EAAT on HR which was significantly reduced on day 2 during the actual EAAT session. Equine-Assisted Activities and Therapies had no effect on respiration rate and systolic or diastolic blood pressure in veterans involved in five sessions of EAAT, lasting 60 minutes in duration over the course of 5 days. Stress levels, as demonstrated by plasma cortisol concentrations and HRV, did not change in horses involved in EAAT sessions with veterans who had been previously diagnosed with PTSD. Furthermore, the horses used in this study did not demonstrate increased levels of well-being as demonstrated by the lack of change in plasma oxytocin concentrations after EAAT sessions. Symptoms of PTSD did change significantly in the veterans who participated in this study.