INTRODUCTION: Testosterone deficiency syndrome (TDS) is characterized by clinical signs of testosterone deficiency in men with reduced testosterone levels. It leads to endothelial dysfunction, which, apart from erectile dysfunction, accelerates atherosclerosis progression. OBJECTIVES: The aim of the study was to evaluate the effect of testosterone supplementation in men with metabolic syndrome (MS) and TDS on autonomic balance assessed by heart rate variability (HRV) in 24鈥慼our Holter monitoring. PATIENTS AND METHODS: The study included 80 men divided into 3 groups: with MS and TDS (MS+TDS+, n = 30), with MS and without TDS (MS+TDS-, n = 25), and healthy controls (n = 25). The MS+TDS+ group received intramuscular testosterone therapy (Omnadren 250) for 9 weeks. Holter monitoring was performed in all patients at baseline and at the end of the therapy. RESULTS: Almost all HRV parameters were significantly lower in the MS+TDS+ group compared with controls. Moreover, total power (TP) as well as high- and low鈥慺requency domains (HF and LF, respectively) were significantly lower in the MS+TDS+ group compared with the MS+TDS- group. There were significant differences in the standard deviation (SD) of normal-to-normal (NN) intervals (SDNN), SDNN index (SDNNI), and SDANN (SD of the averages of NN intervals in all 5鈥憁inute segments) as well as in ultra鈥憀ow鈥慺requency (ULF) domain between the MS+TDS- group and controls. Testosterone supplementation resulted in a statistically significant increase in SDNN, SDANN, TP, LF, ULF, and very鈥憀ow鈥慺requency domain. However, the values did not reach those observed in the control group. The levels of total and free testosterone were not significantly affected by the treatment. CONCLUSIONS: A 9鈥憌eek testosterone supplementation therapy improves HRV parameters. Although these parameters did not reach the values observed in healthy men, the therapy may reduce cardiovascular risk in men with MS and TDS.