Autonomic neuropathy (AN) is a common and serious complication of diabetes. Early detection is essential to enable appropriate interventional therapy. It has long been recognized that subjects with diabetic peripheral neuropathy (DPN) are at much greater risk of developing AN, but there is currently no simple screening tool to assess them. The aim of this study was to investigate pupil responsiveness in diabetic subjects with and without DPN using dynamic pupillometry. During the first test, one flash was administered and the pupil response recorded for 3 seconds. In the second test, twenty-five flashes at one-second intervals were administered and the pupil response recorded for 30 seconds. Several time related parameters were computed from the results. A total of 29 diabetic subjects (17 no DPN, 12 DPN) and 25 healthy volunteers took part in the study. In the first test, pupil-iris ratios in darkness, large deviation and plateau were significantly different between groups. Latency time from flash exposure to the start of constriction was significantly longer in diabetic subjects with DPN compared to healthy volunteers. There was no difference in latency times of largest deviation, plateau or duration of constriction between groups. In the second test, the pupil-iris ratios evaluated in the frame preceding the tenth and the twenty-fifth light flash were significantly greater in healthy volunteers than diabetic subjects with DPN. Latency time from the tenth and twenty-fifth flash exposure to the start of constriction was significantly shorter in healthy volunteers than in diabetic subjects with DPN.