This study looked at spectral analysis of heart rate variability (HRV) between patients with type 2 diabetes mellitus (DM) and healthy controls. The association between diabetic autonomic neuropathy (DAN) with HRV parameter changes and DM risk factors (including nephropathy) was investigated. HRV parameters were compared between 271 patients with DM and 160 controls. A statistically significant difference was found between the two groups for each parameter. Patients with DM were then divided into three groups by the levels of individual risk factors: body mass index, total cholesterol, 2-h postprandial plasma glucose concentration, glycosylated haemoglobin, duration of DM and the albumin-creatinine ratio. HRV parameters decreased significantly in patients with DM as the risk factor level progressed. This study identified previously known and new potential risk factors for the development of DAN, which may be important for the development of risk reduction strategies.