Background: As a side effect of interscalene brachial plexus block (ISBPB), stellate ganglion block (SGB) causes reductions in pupil size (Horner’s syndrome) and cardiac sympathetic nervous activity (CSNA). Reduced CSNA is associated with hemodynamic instability when patients are seated. Therefore, instantaneous measurements of CSNA are important in seated patients presenting with Horner’s syndrome. However, there are no effective tools to measure real-time CSNA intraoperatively. To evaluate the usefulness of pupillometry in measuring CSNA, we investigated the relationship between pupil size and CSNA. Methods: Forty-two patients undergoing right arthroscopic shoulder surgery under ISBPB were analyzed. Pupil diameters were measured at 30 Hz for 2 s using a portable pupillometer. Bilateral pupil diameters and CSNA (natural-log-transformed low-frequency power [0.04–0.15 Hz] of heart rate variability [lnLF]) were measured before ISBPB (pre-ISBPB) and 15 min after transition to the sitting position following ISBPB (post-sitting). Changes in the pupil diameter ([right pupil diameter for post-sitting – left pupil diameter for post-sitting] – [right pupil diameter for pre-ISBPB – left pupil diameter for pre-ISBPB]) and CSNA (lnLF for post-sitting – lnLF for pre-ISBPB) were calculated. Results: Forty-one patients (97.6%) developed Horner’s syndrome. Right pupil diameter and lnLF significantly decreased upon transition to sitting after ISBPB. In the linear regression model (R2 =0.242, P=0.001), a one-unit decrease (1 mm) in the extent of changes in the pupil diameter reduced the extent of changes in lnLF by 0.659 ln(ms2/Hz) (95% CI [0.090, 1.228]). Conclusions Pupillometry is a useful tool to measure changes in CSNA after the transition to sitting following ISBPB.