The primary aim of this study was to assess the retest stability of the short-term heart rate variability (HRV) measurement performed within one session and without the use of any intervention. Additionally, a precise investigation of the possible impact of intrinsic biological variation on HRV reliability was also performed. First, a single test-retest HRV measurement was conducted with 20-30 min apart from one another. Second, the HRV measurement was repeated in ten non-interrupted consecutive intervals. The lowest typical error (CV = 21.1%) was found for the square root of the mean squared differences of successive RR intervals (rMSSD) and the highest for the low frequency power (PLF) (CV = 93.9%). The standardized changes in the mean were trivial to small. The correlation analysis revealed the highest level for ln rMSSD (ICC = 0.87), while ln PLF represented the worst case (ICC = 0.59). The reliability indices for ln rMSSD in 10 consecutive intervals improved (CV = 9.9%; trivial standardized changes in the mean; ICC = 0.96). In conclusion, major differences were found in the reliability level between the HRV indices. The rMSSD demonstrated the highest reliability level. No substantial influence of intrinsic biological variation on the HRV reliability was observed.