Heart rate variability (HRV) is reduced in diabetes mellitus (DM) patients, suggesting dysfunctionof cardiac autonomic regulation and an increased risk for cardiac events. The aimof this paper was to examine the associations of blood glucose level (BGL), glycated hemoglobin(HbA1c), and duration of diabetes with cardiac autonomic regulation assessed byHRV analysis. Resting electrocardiogram (ECG), recorded over 20 min in supine position,and clinical measurements of 189 healthy controls and 93 type 2 DM (T2DM) patients wereanalyzed. HRV was assessed using several time-domain, frequency-domain, and non-linearmethods. HRV parameters showed a clear difference between healthy controls andT2DMpatients. Hyperglycemia was associated with increase in mean heart rate and decreasein HRV, indicated by negative correlations of BGL and HbA1c with mean RR interval andmost of the HRV parameters. Duration of diabetes was strongly associated with decreasein HRV, the most significant decrease in HRV was found within the first 5'10 years of thedisease. In conclusion, elevated blood glucose levels have an unfavorable effect on cardiacautonomic function and this effect is pronounced in long-term T2DM patients. The mostsignificant decrease in HRV related to diabetes and thus presence of autonomic neuropathywas observed within the first 5'10 years of disease progression.