This study was designed to investigate the question of whether psychophysiological changes during EMDR sessions are related to subjective and objective reduction of PTSD symptoms. During-session changes in autonomic tone in relation to session-to-session changes of subjective stress, trauma-related symptoms, and psychophysiological reactions during a traumatic reminder were investigated in 10 patients suffering from single-trauma PTSD. Treatment duration followed each patient’s individual needs and ranged between 1 and 4 sessions, resulting in a total of 24 EMDR treatment sessions from which psychophysiological data were completely recorded. Treatment with EMDR was followed by a significant reduction of trauma-related symptoms, elimination of the PTSD diagnosis in 8 of the 10 participants, as well as by significantly reduced psychophysiological reactivity to an individualized trauma script. Psychophysiological dearousal in sessions correlated significantly with decrease in script-related reactions in heart rate and parasympathetic tone, and with changes in subjective disturbance. Our results indicate that information processing during EMDR is followed by during-session decrease in psychophysiological activity, reduced subjective disturbance and reduced stress reactivity to traumatic memory.