Even though various research has examined the factors that cause visual discomfort in watching stereoscopic 3D video, the brightness factor has not been dealt with sufficiently. In this paper, we analyze visual discomfort under various illumination conditions by considering eye-blinking rate and saccadic eye movement. In addition, we measure the perceived depth before and after watching 3D stereoscopic video by using our own 3D depth measurement instruments. Our test sequences consist of six illumination conditions for background. The illumination is changed from bright to dark or vice-versa, while the illumination of the foreground object is constant. Our test procedure is as follows: First, the subjects are rested until a baseline of no visual discomfort is established. Then, the subjects answer six questions to check their subjective pre-stimulus discomfort level. Next, we measure perceived depth for each subject, and the subjects watch 30-minute stereoscopic 3D or 2D video clips in random order. We measured eye-blinking and saccadic movements of the subject using an eye-tracking device. Then, we measured perceived depth for each subject again to detect any changes in depth perception. We also checked the subject’s post-stimulus discomfort level, and measured the perceived depth after a 40-minute post-experiment resting period to measure recovery levels. After 40 minutes, most subjects returned to normal levels of depth perception. From our experiments, we found that eye-blinking rates were higher with a dark to light video progression than vice-versa. Saccadic eye movements were a lower with a dark to light video progression than viceversa.