OBJECTIVE: Pathophysiology explaining pain in diabetic neuropathy (DN) is still unknown. RESEARCH DESIGN AND METHODS: Thirty patients with peripheral DN (17 men and 13 women; mean age 52.4 +/- 2.5 years) were investigated. Fifteen patients had neuropathic pain, and 15 patients were free of pain. Patients were followed over 2 years and examined at the beginning and thereafter every 6 months. Clinical severity and painfulness of the DN were assessed by the neuropathy impairment score and visual analog scales (VASs). Cold and warm perception thresholds as well as heat pain thresholds were obtained for evaluation of Adelta- and C-fibers. Nerve conduction velocities (NCVs) and vibratory thresholds were recorded for analysis of thickly myelinated fibers. Moreover, for assessment of cardiac vagal function, heart rate variability (HRV) was evaluated. In order to reduce day-to-day variability of pain, mean values of the five time points over 2 years were calculated and used for further analysis. Data were compared with an age- and sex-matched control group of healthy volunteers. RESULTS: There were significant differences regarding electrophysiological studies, HRV and quantitative sensory testing (QST) between patients and healthy control subjects (P < 0.001). Generally, patients with neuropathic pain were indistinguishable from pain-free patients. In the pain group, however, VAS pain ratings were correlated to the impairment of small-fiber function (cold detection thresholds, P = 0.02; warm detection thresholds, P = 0.056). CONCLUSIONS: Intensity of pain in painful DN seems to depend on small nerve fiber damage and deafferentation.