Introduction: The purpose of this study was to develop and initially validate an objective, yet quick, method for assessment of human fatigue. This aim can be achieved by the use of an optical, non-contact method of analysis of pupil activity. The study involved 2 phenomena typical for the behavior of the human pupil: pupillary reflex and pupillary movements. The 1st phenomenon is related to the pupillary light reflex (PLR), which presents the results of physiological adaptation mechanisms of the human eye. The pupillary unrest index (PUI) is an additional parameter referring to the tendency for instability of the pupil. Material and methods: Indicators of these 2 mechanisms were assessed under the same experimental laboratory conditions. Assessment was conducted on a group of volunteers (N = 10) during 4 controlled series of measurements performed at night. Pupillary reflex parameters associated with PLR and pupillary unrest index (PUI) were recorded using F²D Fit-For-Duty, a commercially available system made by AMTech Pupilknowlogy GmbH. Baseline pupil diameter, oscillations, reflex latency, maximum reaction time, pupil constriction time, pupil dilation time, and constriction amplitude were recorded. Results: As a result of the study, we were able to demonstrate correlation and confirm the usefulness of PLR and PST methods in the assessment of sleep deprivation. Parameters of PLR that may indicate human fatigue were identified. The effect of light impulse sequences on the dynamics of pupillary reflex and the relationship between PUI (pupillary unrest index) measurement duration and sleepiness assessment validity, were assessed. Conclusions: The results of the pilot studies were sufficient to develop minimum requirements for a PLR sensor that would be capable of estimating the level of fatigue with accuracy of a PUI method, but at a 5 times faster rate.