Eye movement desensitization and reprocessing (EMDR) is an established treatment for post-traumatic stress disorder (PTSD). However, its working mechanism remains unclear. This study explored physiological correlates of eye movements during EMDR in relation to current hypotheses; distraction, conditioning, orienting response activation, and REM-like mechanisms. During EMDR therapy, fingertip temperature, heart rate, skin conductance, expiratory carbon dioxide level, and blood pulse oximeter oxygen saturation, were measured in male subjects with PTSD. The ratio between the low and high frequency components of the heart rate power spectrum (LF/HF) were computed as measures of autonomic balance. Respiratory rate was calculated from the carbon dioxide trace. Stimulation shifted the autonomic balance as indicated by decreases in heart rate, skin conductance and LF/HF-ratio, and an increased finger temperature. The breathing frequency and end-tidal carbon dioxide increased; oxygen saturation decreased during eye movements. In conclusion, eye movements during EMDR activate cholinergic and inhibit sympathetic systems. The reactivity has similarities with the pattern during REM-sleep.