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The problem of eyestrain caused by 3D display becomes a major topic of discussion. However, there has been no previous research to compare the eyestrain caused by 2D and 3D displays, objectively. In this study, we measured the frequency of blinking using a near-infrared pupil detection device to quantitatively compare the amount of eyestrain caused when watching the two types of displays, on the basis that more frequent blinking indicates greater eyestrain. Our research is novel in the following three ways. First, the eyestrain that occurs when watching 2D and 3D displays are quantitatively compared by using eye blink frequency. Second, the significances of two factors (kind of display, and the Z distance of watching display) and their interaction effect are calculated by using 2k factorial design analysis. Third, the correlation between objective tests based on blink frequency and subjective tests based on a survey is measured. The experimental results showed that the amount of eyestrain when watching a 3D display, especially at short viewing distances, was higher. High correlation between the quantitative results and subjective assessments collected using a questionnaire survey showed that blinking frequency is a reliable measure. Further, the developed pupil tracking device and blinking-rate-based measurement can be easily adapted to commercial glasses-based 3D display systems.