Since heart rate variability (HRV) is associated with average heart rate (HR) and respiratory rate (RespRate), alterations in these parameters may impose changes in HRV. Hence the repeatability of HRV measurements may be affected by differences in HR and RespRate. The study aimed to evaluate HRV repeatability and its association with changes in HR and RespRate.Forty healthy volunteers underwent two ECG examinations 7 days apart. Standard HRV indices were calculated from 5-min ECG recordings. The ECG-derived respiration signal was estimated to assess RespRate. To investigate HR impact on HRV, HRV parameters were corrected for prevailing HR.Differences in HRV parameters between the measurements were associated with the changes in HR and RespRate. However, in multiple regression analysis only HR alteration proved to be independent determinant of the HRV differences-every change in HR by 1 bpm changed HRV values by 16.5% on average. After overall removal of HR impact on HRV, coefficients of variation of the HRV parameters significantly dropped on average by 26.8% (p < 0.001), i.e., by the same extent HRV reproducibility improved. Additionally, the HRV correction for HR decreased association between RespRate and HRV.In stable conditions, HR but not RespRate is the most powerful factor determining HRV reproducibility and even a minimal change of HR may considerably alter HRV. However, the removal of HR impact may significantly improve HRV repeatability. The association between HRV and RespRate seems to be, at least in part, HR dependent.