Different levels of visual fatigue in the human eye depend on different color-formation methods and image quality. This paper uses the high-frequency component of the spectral power of accommodative microfluctuations as a major objective indicator for analyzing the effects of visual fatigue based on various displays, such as color-formation displays and 3D displays. Also, a questionnaire is used as a subjective indicator. The results are that 3D videos cause greater visual fatigue than 2D videos (p < 0.001), the shutter-type 3D display causes visual fatigue more than the polarized type (p = 0.012), the display of the time-sharing method causes greater visual fatigue than the spatial-formation method (p = 0.008), and there is no significance between various light source modules of displays (p = 0.162). In general, people with normal color discrimination have more visual fatigue than those with good color discrimination (p < 0.001). Therefore, this paper uses the high-frequency component of accommodative microfluctuations to evaluate the physiological stress or strain by overexerting the visual system, and can compare the level of visual fatigue between various displays.