This study was conducted to assess the relationship between fatigue and pupillary responses. Pupillary responses, ECG and blood pressure were measured for 24 hours every 30 min in 8 subjects. A questionnaire was used to rate subjective feeling of fatigue. Twenty-four hours were divided equally into four 6-hour blocks. Subjective feeling of fatigue increased markedly in the fourth block, and the difference in subjective fatigue between fourth and first blocks was significant. Of nine pupillary responses, the pupil diameter was found to decrease with time. With respect to the function of the autonomic nervous system such as heart rate, systolic blood pressure and diastolic blood pressure, only heart rate was found to be sensitive to the increased subjective feeling of fatigue. A significant difference was found in the mean pupil diameter and mean heart rate between the last and first blocks. This result indicates that pupil diameter is related to fatigue and can be used to assess fatigue. Possible implications for fatigue assessment are discussed.