Easy-to-use and accurate heart rate variability (HRV) assessments are essential in athletes’ follow-up, but artifacts may lead to erroneous analysis. Artifact detection and correction are the purpose of extensive literature and implemented in dedicated analysis programs. However, the effects of number and/or magnitude of artifacts on various time- or frequency-domain parameters remain unclear. The purpose of this study was to assess the effects of artifacts on HRV parameters. Root mean square of the successive differences (RMSSD), standard deviation of the normal to normal inter beat intervals (SDNN), power in the low- (LF) and high-frequency band (HF) were computed from two 4-min RR recordings in 178 participants in both supine and standing positions, respectively. RRs were modified by (1) randomly adding or subtracting 10, 30, 50 or 100 ms to the successive RRs; (2) a single artifact was manually inserted; (3) artifacts were automatically corrected from signal naturally containing artifacts. Finally, RR recordings were analyzed before and after automatic detection-correction of artifacts. Modifying each RR by 10, 30, 50 and 100 ms randomly did not significantly change HRV parameters (range -6%, +6%, supine). In contrast, by adding a single artifact, RMSSD increased by 413% and 269%, SDNN by 54% and 47% in supine and standing positions, respectively. LF and HF changed only between -3% and +8% (supine and standing) in the artifact condition. When more than 0.9% of the signal contained artifacts, RMSSD was significantly biased, whilst when more than 1.4% of the signal contained artifacts LF and HF were significantly biased. RMSSD and SDNN were more sensitive to a single artifact than LF and HF. This indicates that, when using RMSSD only, a single artifact may induce erroneous interpretation of HRV. Therefore, we recommend using both time- and frequency-domain parameters to minimize the errors in the diagnoses of health status or fatigue in athletes.