Previous research has indicated that viewing 3D displays may induce greater visual fatigue than viewing 2D displays. Whether viewing 3D displays can evoke measureable emotional responses, however, is uncertain. In the present study, we examined autonomic nervous system responses in subjects viewing 2D or 3D displays. Autonomic responses were quantified in each subject by heart rate, galvanic skin response, and skin temperature. Viewers of both 2D and 3D displays showed strong positive correlations with heart rate, which indicated little differences between groups. In contrast, galvanic skin response and skin temperature showed weak positive correlations with average difference between viewing 2D and 3D. We suggest that galvanic skin response and skin temperature can be used to measure and compare autonomic nervous responses in subjects viewing 2D and 3D displays.