Both diabetes mellitus (DM) and the metabolic syndrome (MetS) are associated with autonomic neuropathy, which predisposes to cardiac events and death. Measures of heart rate variability (HRV) can be used to monitor the activity of the autonomic nervous system (ANS), and there are strong indications that HRV can be used to study the progression of ANS-related diabetes complications. This study aims to investigate differences in HRV in healthy, MetS and diabetic populations. Based on 7880 participants from the sixth health survey in Tromsø (Tromsø 6, 2007-2008), we found a significant negative association between the number of MetS components and HRV as estimated from short-term pulse wave signals (PRV). This decrease in PRV did not appear to be linear, instead it leveled off after the third component, with no significant difference in PRV between the MetS and DM populations. There was a significant negative association between HbA1c and PRV, showing a decrease in PRV occurring already within the normal HbA1c range. The MetS and DM populations are different from healthy controls with respect to PRV, indicating impaired ANS in both conditions. In the future, a study with assessment of PRV measurements in relation to prospective cardiovascular events seems justified.