Abstract Background The measurement of the pupillary function is an indispensable test in some eye examinations, being necessary the evaluation of the precision of instruments performing such measures. The aim of this study was to evaluate the intrasession repeatability of pupil size measurements provided by a multidiagnostic platform in a large sample of healthy eyes. Methods This prospective study enrolled 100 healthy eyes of 100 patients, with ages ranging from 23 to 65 years old. Repeated pupil size measures under photopic (P, 220 lx), mesopic (M, 160 lx), low mesopic (L, 70 lx), and scotopic conditions (S, 1 lx) were obtained with the VX120 system (Visionix-Luneau Technologies, Chartres, France) after a complete eye exam. Likewise, pupil size was also measured once in the fellow eye in a total of 75 eyes. The level of intrasession variability as well as differences between fellow eyes were evaluated. Results Most of differences between repeated measures did not exceed 0.5 mm (82% of S and 100% of P below this value). No significant differences between these repeated measures were found for S (p = 0.099) and L (p = 0.751). However, statistically significant differences were found between repeated measures for M (p = 0.002) and P (p = 0.003). The analysis of clinical relevance of differences between pairs (Passing-Bablok) only confirmed the clinical relevance of differences between the first and second repeated measurement of M. Concerning the comparative analysis between fellow eyes, no statistically significant differences in pupil size were found between right and left eyes in any light condition evaluated (p ≥ 0.227). Conclusions The VX120 system can provide consistent measurements of pupil size under scotopic, low mesopic and photopic conditions, with a relative limitation under mesopic conditions.