This study aimed to determine whether online-guided mindfulness-based interventions (MBIs) in daily life improve autonomic nervous system function as measured by the pupillary light reflex (PLR) in healthy subjects. A total of 94 university students were randomly allocated to either an intervention group or a waitlist control group. Participants practiced single-session meditation in a laboratory. During the intervention period, the participants practiced brief online mindfulness meditation for 12 weeks. PLR and psychological indices were measured before and after single and multiple mindfulness sessions. Using a multiple-regression analysis controlling for the baseline values, participants in the intervention group showed significantly decreased emotional fluctuations and significantly increased relaxation compared to those in the control group in a single session, whereas no significant differences in physiological indices were detected. In 12-week multiple sessions, participants in the intervention group showed significantly decreased state anxiety (p = 0.05), and marginally significantly decreased sympathetic nervous system activation (VD, p = 0.08) and perceived stress (p = 0.07) compared to those in the control group. These results suggest that the effects of mindfulness meditation on autonomic nerve function may be cumulative. These findings highlight the preventive effects of continuing online-guided MBIs in a healthy population and the potential usefulness of pupilometers in monitoring intervention effects. The applicability of pupillometry is further supported by the finding that MBIs improved psychological indices. Trial Registration: This study was not pre-registered.