Stereoscopic 3D is undoubtedly one of the most attractive content. It has been deployed intensively during the last decade through movies and games. Among the advantages of 3D are the strong involvement of viewers and the increased feeling of presence. However, the sanitary e ects that can be generated by 3D are still not precisely known. For example, visual fatigue and visual discomfort are among symptoms that an observer may feel. In this paper, we propose an investigation of visual fatigue generated by 3D video watching, with the help of eye-tracking. From one side, a questionnaire, with the most frequent symptoms linked with 3D, is used in order to measure their variation over time. From the other side, visual characteristics such as pupil diameter, eye movements ( xations and saccades) and eye blinking have been explored thanks to data provided by the eye-tracker. The statistical analysis showed an important link between blinking duration and number of saccades with visual fatigue while pupil diameter and xations are not precise enough and are highly dependent on content. Finally, time and content play an important role in the growth of visual fatigue due to 3D watching. © (2013) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).