The acoustic startle reflex can be modulated by positive and negative emotion. There is evidence that this modulation can be influenced by voluntary attempts to regulate emotion, and that startle modulation during emotion regulation is more reflective of changes in arousal than valence. However, whether valence and arousal play similar roles in emotion regulation across different psychophysiological indices is unclear. The goal of this study was to characterize further the relative contributions of valence and arousal to changes in psychophysiological responsiveness during voluntary emotion regulation, using multiple psychophysiological measures including eyeblink startle, skin conductance, and heart rate. We studied 10 healthy adults, and found that voluntary attempts to down-regulate positive and negative emotion resulted in decreased eyeblink startle magnitude, skin conductance responses, and heart rate, relative to attempts to up-regulate emotion. These findings indicate that the volitional regulation of emotion had systematic effects on psychophysiological parameters which were similar for positive and negative emotion, suggesting that psychophysiological responsiveness during emotion regulation is more strongly influenced by the modulation of arousal than by the valence of the regulated emotion.