It has been shown that impaired cardiac autonomic activity is closely related with lethal arhythmias. Heart rate variability (HRV), analysis of beat-to-beat variations, is an important and widely used non-invasive method to assess autonomic function. Impaired cardiac autonomic activity and altered sympathovagal balance were previously documented in patients with hypothyroidism. However, the effect of subclinical hypothyroidism (SH) on autonomic function has not been studied yet. We aimed to investigate the effect of SH on sympathovagal balance using the HRV method. The study included 31 patients with SH and 28 healthy volunteer controls. Patients with cardiac, metabolic, neurological disease or any other systemic disease that could affect autonomic activity were excluded from the study. HRV time domain and frequency domain parameters were determined over a period of 24 h. All time and frequency domain measures of HRV in patients with SH were not significantly different compared to those of healthy control group (p > 0.05). Additionally, we compared SH subgroups (TSH level > or =10 and TSH level <10 mU/l) with each other and the controls. A statistically significant difference was observed only in time domain parameters of SD of normal-to-normal intervals (SDNN) and SD of all 5-min mean normal NN intervals (SDANN) between subgroup with TSH level > or =10 and controls (p < 0.05, p < 0.05, respectively). In correlation analysis with TSH, there was positive relationship between TSH and the root mean square of successive differences between adjacent R-R intervals (rMSSD). These findings indicate that SH may affect cardiac autonomic activity in correlation with TSH levels.