Objectives: The pupillary light reflex (PLR), pupillary sleepiness test (PST), and visual evoked potentials (VEPs) are crucial for assessing the functional integrity of the visual and autonomic nervous systems. These responses integrate retinal phototransduction with midbrain reflex pathways and cortical visual processing, all of which are influenced by physiological and clinical disorders. Their temporal dynamics lay the groundwork for using pupillary reflexes and cortical potentials as biomarkers of fatigue and the effects of psychoactive substances on them. This study aimed to identify physiological biomarkers of fatigue by evaluating the PLR and VEPs. Material and Methods: These modalities were validated against the PST, pupillary unrest index parameters, and blink-rate analysis to assess autonomic and cortical contributions to fatigue. This study examined the effects of 48-hour sleep deprivation on pupillary behavior in 18 healthy adults under controlled conditions. Results: Analysis of PLR dynamics revealed that the dilation-phase parameters D_Dil1 and D_Dil2, corresponding to pupil diameters at 20% and 80% recovery, respectively, were sensitive to changes in autonomic activation. These complemented the established fatigue indices, including constriction velocity, relative amplitude, time of minimum diameter, and latency. Visual evoked potentials analysis demonstrated that sustained wakefulness modulated cortical responses, with a delay and amplitude reduction in the N145 component. Conclusions: Biomarkers from the pupillary reflex dilation phase and N145 LAT in VEP recordings serve as reliable indicators of fatigue severity, as corroborated by the PST and Stanford Sleepiness Scale assessments. Int J Occup Med Environ Health. 2026;39(5)