OBJECTIVE Although a clear link between diabetic peripheral neuropathy (DPN) and autonomic neuropathy is recognized, the relationship of autonomic neuropathy with subtypes of DPN is less clear. This study aimed to investigate the relationship of autonomic neuropathy with painless and painful DPN. METHODS Eighty subjects (20 healthy volunteers, 20 with no DPN, 20 with painful DPN, 20 with painless DPN) underwent detailed neurophysiological investigations (including conventional autonomic function tests [AFTs]) and spectral analysis of short-term heart rate variability (HRV), which assesses sympathovagal modulation of the heart rate. Various frequency-domain (including low frequency [LF], high frequency [HF], and total power [TP]) and time-domain (standard deviation of all normal-to-normal R-R intervals [SDNN] and root mean square of successive differences [RMSSD]) parameters were assessed. RESULTS HRV analysis revealed significant differences across the groups in LF, HF, TP, SDNN, and RMSSD (ANOVA P < 0.001). Subgroup analysis showed that compared with painless DPN, painful DPN had significantly lower HF (3.59 +/- 1.08 [means +/- SD] vs. 2.67 +/- 1.56), TP (5.73 +/- 1.28 vs. 4.79 +/- 1.51), and SDNN (2.91 +/- 0.65 vs. 1.62 +/- 3.5), P < 0.05. No significant differences were seen between painless DPN and painful DPN using an AFT. CONCLUSIONS This study shows that painful DPN is associated with significantly greater autonomic dysfunction than painless DPN. These changes are only detected using spectral analysis of HRV (a simple test based on a 5-min electrocardiogram recording), suggesting that it is a more sensitive tool to detect autonomic dysfunction, which is still under-detected in people with diabetes. The greater autonomic dysfunction seen in painful DPN may reflect more predominant small fiber involvement and adds to the growing evidence of its role in the pathophysiology of painful DPN.