This study evaluated the time-on-task effect of a sustained attention task on the accommodative response, explored the link between ocular accommodation, cardiac autonomic regulation and behavioral performance, and tested the inter-session repeatability of these results by performing the same experimental procedure on different days. The accommodative response was measured in 25 university students using a WAM-5500 autorefractor, while participants performed a 10-minute psychomotor vigilance task at 50 cm. There were no time-on-task effects for the lag of accommodation, whereas the microfluctuations of accommodation and reaction time were modulated as a function of time-on-task, observing a progressive increment of both variables over time. Multiple regression analysis revealed that the microfluctuations of accommodation were a reasonably good predictor of behavioral performance. Analysis of inter-session repeatability showed that ocular accommodation, heart rate variability and reaction time had a low to moderate level of repeatability between two measurements. The microfluctuations of accommodation were sensitive to time-on-task effects during sustained attention, with a lower stability of accommodation being linked to reduced behavioral performance. The predictive capacity of the variability of accommodation for behavioral performance might be explained by the brain mechanisms shared by the ocular dynamics and attentional state; however, future studies would be required to elucidate this association. The low to moderate inter-session repeatability indicated that results in two measurement periods cannot be considered interchangeable, and therefore, the results of this study should be interpreted cautiously in this regard.