This study assessed physiological measures for the study of emotional dysregulation associated with borderline personality disorder (BPD). Two patient groups, the first comprised of individuals with BPD only (n = 16) and the second, individuals with BPD and co-occurring substance-use disorder (SUD; n = 35), and a group of healthy controls (n = 45) were shown standardized pictures of varying valance and arousal levels. Affective modification of startle eye-blink responses, heart rate, facial electromyography (EMG, including corrugator and zygomatic activity), and skin-conductance responses were collected during picture presentation and during a brief recovery period. Startle data during picture presentation indicated a trend toward the expected increase in startle response magnitude to negative stimuli, to be moderated by group status, with patients with BPD-SUD showing a lack of affective modification and the BPD-only group showing similar affective modification to that of controls. Heart-rate data suggested lower reactivity to negative pictures for both patient groups. Differences in facial EMG responses did not provide a clear pattern, and skin-conductance responses were not significantly different between groups. The data did not suggest differences between groups in recovery from exposure to the emotional stimuli. The startle and heart-rate data suggest a possible hyporeactivity to emotional stimuli in BPD.