Heart rate variability (HRV) is becoming one of the most commonly used tools for tracking the time course of training adaptation/maladaptation of athletes and setting optimal training loads, leading to improved performance. The aim of this study was to compare Realtrack Systems manufacturer software (sPRO) with the Kubios HRV Standard. R-R intervals were recorded from 48 athletes using a chest strap synchronized to WIMU PRO™ device for 10 min to analyze time-domain HRV indices, located between the scapulae. Time-domain HRV indices analyzed included the square root of the mean squared differences of successive R-R intervals (RMSSD), standard deviation of normal R-R intervals (SDNN), and percentage of adjacent R-R intervals that differ from each other by more than 50 ms (pNN50). The intraclass correlation test and Bland-Altman plot were used to verify the agreement between the sPRO and the Kubios HRV Standard software for examining time-domain HRV indices. The intraclass correlation coefficient showed high values of agreement of R-R intervals and time-domain HRV indices between the two software options (RMSSD, SDNN, and pNN50; ICC > 0.951). The Bland-Altman plot showed low bias for all the HRV indices analyzed (bias = −0.11 to −1.08). In addition, no systematic bias was found between the residual of the mean difference and the average values of the two software packages ( p > 0.05). Therefore, both software options can be used interchangeably for analyzing time-domain HRV records.