Corneal confocal microscopy (CCM) derived corneal nerve measures are lower in diabetic sensorimotor polyneuropathy (DSPN). There are, however, methodological challenges in relation to adequate and unbiased sampling of images with objective corneal nerve quantification. Here we compare a new sampling method and adjusted area calculation with established methods of corneal nerve quantification in patients with and without DSPN and healthy controls. CCM images from 26 control subjects and 62 patients with type 1 diabetes with (n = 17) and without (n = 45) DSPN were analyzed. The images were randomly selected and corneal nerve fiber length (CNFL), corneal nerve fiber branch density (CNBD) and corneal nerve fiber density (CNFD) were determined in both a manual and automated manner. The new method generated 8-40% larger corneal nerve parameters compared to the standard procedure (p < 0.05). CNFL was significantly reduced using the new method for both manual and automated analysis; whilst CNFD and CNBD were significantly reduced using the automated method in both diabetic groups compared with controls. The new, objective method showed a reduction in corneal nerve parameters in diabetic patients with and without DSPN. We recommend using a randomized sampling method and area-dependent analysis to enable objective unbiased corneal nerve quantification.