In this paper, a subjective study is presented which aims to measure the minimum perceivable depth difference on an autostereoscopic display in order to provide an indication for visual fatigue. The developed experimental setup was used to compare the subject's performance before and after 3D excitation on an autostereoscopic display. By comparing the results to a verification session with 2D excitation, the effect of 3D visual fatigue can be isolated. It was seen that it is possible to reach the threshold of acuity for stereo disparity on that autostereoscopic display. It was also found that the measured depth acuity is slightly higher after 3D viewing than after 2D viewing.