Many intervention studies assume the stability of heart rate variability (HRV) parameters, and their sample sizes are often small, which can significantly affect their conclusions. The aim of this study is to assess the stability and reliability of standard HRV parameters within a single resting session, and to estimate the sample size required to detect the minimal clinically important effect of an intervention. Heart rate was recorded in 50 adult healthy subjects for 50 min in a seated position. Eight standard HRV parameters were calculated from five evenly spaced 5 min intervals. Stability was assessed by comparing the mean values of HRV parameters between the consecutive five test–retest measurements. Absolute reliability was determined by standard error of measurement, and relative reliability by intraclass correlation coefficient. The sample size required to detect a mean difference of ≥30% of between-subject standard deviation was estimated. As expected, almost all HRV parameters had poor absolute reliability but most HRV parameters had substantial to excellent relative reliability. We found statistically significant differences in almost all HRV parameters between the first 20 min and the last 30 min of the session. The estimated sample size ranged from 19 to 300 subjects for the first 20 min and from 36 to 194 subjects for the last 30 min of the session, depending on the selected HRV parameter. We concluded that optimal HRV measurement protocols in a resting seated position should be performed within the first 20 min or between 20 and 50 min after assuming a resting seated position. Future interventional HRV studies should include a sufficient number of subjects and consider the Bonferroni correction according to the number of selected HRV parameters to achieve an appropriate level of study power and precision.