OBJECTIVES: Dynamic pupillometry is a simple screening tool for quantifying pupillary light reflex (PLR), to indicate autonomic nervous system (ANS) activity. Heart rate variability (HRV) is the gold standard method for assessing ANS effects on the heart. The present study aimed to compare ANS activity as measured using dynamic pupillometry (DP) with short term HRV in healthy volunteers. METHODS: The study includes 200 participants aged between 20-60 years. PLR was measured using infrared videography and categorized into different quantitative parameters that reflect ANS activity. Simultaneously, Lead II ECG was recorded for 5鈥痬in to evaluate the short term HRV of time and frequency domain parameters. The data obtained from the two methods were compared with each other to provide the relationship between PLR and HRV. RESULTS: Study participants\' mean age was 36.95鈥?/-鈥?.45 years. The different pupillary indices of PLR and all the HRV parameters were within the range of normative data. Several PLR indicators of the parasympathetic function include mean constriction velocity (MCV; r鈥?鈥?.60, P鈥?鈥?.001), absolute constriction amplitude (ACA; r鈥?鈥?.57, P鈥?鈥?.001) baseline pupil diameter (BPD; r鈥?鈥?.44, p鈥?鈥?.001), and minimum pupil diameter (MPD; r鈥?鈥?0.35, p鈥?鈥?.001) were significantly correlated with high-frequency power (HF in normalized units) of HRV signal. CONCLUSION: PLR variables showed a significant relationship to HRV indices. Dynamic pupillometry can be a complementary tool to HRV for evaluating ANS activity.