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This study explored the association between pupil area variation, heart rate variability, and fatigue, caused by alcohol consumption. Sixteen healthy young male volunteers were enrolled in this study for a series of structured physical examinations. Individual drinking volume was strictly assessed in accordance with the normalized experimental design. The motions of the subjects' pupils, as well as electrocardiograms were recorded by a computerized recording system, both before and after drinking alcohol. After alcohol intake, the pupil diameter, and both low frequency and standard deviation of all heartbeat interval indices of heart rate variability showed significant variations compared with the baseline. In addition, time- and frequency-domain indices of heart rate variability were negatively associated with pupil area. Thus the current study suggests that fatigue status may be determined by significant changes in pupil area and heart rate variability.