With the development of 3D display technology, the effect of 3D display attracts more and more attention. Vertical parallax is one of main factors that cause visual fatigue, and its evaluation methods are all subjective. In this paper, we investigate the use of electroencephalography (EEG) as a tool to evaluate vertical parallax, measuring the EEG response signal by presenting a series levels of vertical parallax images. Experimental results show that presence of vertical parallax reliably elicits a measurable response in the brain. We furthermore show that the reaction varies depending on the vertical parallax levels, therefore, EEG can be used to evaluate the effect of vertical parallax on asthenopia when viewing 3D context. For comfortable viewing content, the vertical parallax should be less than 20-pixels in the best viewing distance.