Several years ago, a famous accident occurred in Japan. Hundreds of children, who were watching a cartoon on television, suddenly complained of spasms and vertigo, and were taken to hospital. In this study, the autonomic nervous system was evaluated during audiovisual stimulation with three dimensional Virtual Reality (VR) imaging. In our previous studies, we designed the diagnosis machine for an autonomic function using multi-parameters, including electrocardiography, arterial blood pressure, respiration and stroke volume as detected by ultrasonic cardiography. Healthy adult volunteers were used in this experiment with their satisfactory informed consent. The three-dimensional content for VR included dinosaur images in a pre-historic scene. The content was projected on a wide screen and volunteers watched an audiovisual screen for about 20 minutes and the 3-D and 2-D images were compared. There was no significant arrhythmia during experiments in both images. No significant alteration was observed in the quantified hemodynamic data during the experiment. Spectral analysis was performed to evaluate the heart rate variability (HRV) during the experiment. LF, HF and LF/HF of HRV were calculated. However, there were no significant changes during the experiment. Significant change was observed in the fractal dimension of the stroke volume during 2-D and 3-D image VR immersion. Our results suggest that a significant response was observed in the autonomic function according to the 2-D or 3-D images. Our study, which aims at safe audiovisual stimulating equipment, must be developed.