Objective. Establishing objective indicators of subjective pain intensity is important in pain assessment. Pupillary light reflex (PLR) and heart rate variability (HRV) indicate autonomic nervous system (ANS) activity and may serve as pain indicators because pain can affect ANS activity. In this prospective longitudinal study, we aimed to investigate the potential of PLR/HRV parameters as objective indicators of subjective pain intensity after tonsillectomy. Approach. Sixty-seven patients undergoing tonsillectomy were enrolled. Subjective pain intensity based on a numeric rating scale (NRS) and eight PLR/HRV parameters were assessed at five time points. We investigated the changes in the NRS values over time. We estimated regression coefficients reflecting parameter changes per unit change in the NRS score using linear mixed-effects models. Main Results. The mean NRS score was 0 at two pre-surgery time points, 5 on postoperative days (PODs) 1 and 2, and 0 at postoperative week 3. Two parameters (initial pupil size [INIT] and constriction latency [LAT]) showed significant changes on POD1 and POD2 in comparison to baseline data measured at the pre-surgery time point. Among these parameters, only LAT showed no significant changes between POD1 and POD2. Significant regression coefficients with the narrowest 95% confidence intervals were observed for INIT and LAT. Increased NRS scores were associated with decreased INIT and shortened LAT. Significance. LAT was a robust indicator of subjective pain intensity. Our patients showed decreased INIT with increased NRS scores, indicating the predominance of the parasympathetic, not sympathetic, tone in pupils. Further studies are required to investigate factors causing this predominance.