Three physiological factors (melatonin levels, tear mucus ferning quality and degree of asthenopia) were examined for their relationship to visual comfort. A lighting environment was created where the illuminance, illuminance uniformity and correlated colour temperature could be adjusted. A three-factor and three-level orthogonal experiment with 24 subjects was designed and carried out. The results indicated that the selected environmental factors had different impacts on the physiological factors. With the illuminance increasing, the melatonin level decreased significantly and the tear mucus ferning quality was improved. However, there is no general influence of illuminance uniformity and correlated colour temperature on the physiological parameters, only differential effects among the three levels were found.