Pain leads to activation of the autonomic nervous system and thus, among other things, to pupillary reflex dilation (PRD). Previous studies have already confirmed a correlation between the perception of pain and the pupillary reaction, measured using pupillometry. However, the previous study populations were under the influence of medication for analgesia in perioperative setting or suffered from pain. This study examines the relationship between pupillary reaction and pain perception in healthy controls and addresses the question of whether endogenous pain inhibition, clinically tested by conditioned pain modulation (CPM), can be quantified using pupillometry. Forty-two healthy volunteers (21 females, 21 males, mean age 27.9 ± 5.8 years, range 20-39 years) were included in this study. The PRD, as a measure of the pupillary reaction (variance from the base diameter in percent), was investigated during baseline, heat application and during CPM testing and results compared to the reported pain intensity on the numerical rating scale (NRS). The volunteers showed higher variances under painful conditions compared to the measurement at rest corresponding to higher sympathetic activity during pain. Volunteers with a higher variance, ie a stronger pupillary reaction, gave higher pain ratings than subjects with a lower pupil variance. However, there was no correlation between the NRS and PRD. PRD and pain ratings during CPM were significantly lower compared to heat pain application alone. However, there was no correlation between the calculated CPM effect and the PRD. Pupillometry is capable of objectively reflecting the pain response, eg pain relief through CPM testing. However, the CPM effect calculated from the subjective pain ratings and the objective PRD measurements is not associated suggesting that both measure different aspects of pain perception. It must be discussed whether the CPM effect can be the correct measure for the functionality of the pain system.