The effect of Stroop test (ST)-induced stress on autonomic nervous system activity is often examined via measurement of heart rate variability (HRV). However, HRV may be significantly affected by interfering factors, including vocalization and inappropriate body position. Surprisingly, published studies ignore these issues, so the aim of our study was to test the innovative procedure for correct HRV measurement in individuals exposed to ST. Moreover, we examined possible gender differences in HRV and heart rate (HR) during ST. Healthy participants (21 men, 34 women) were placed in a supine position (no orthostatic activation) and then exposed to a 5 min relaxation period and a 5 min period of computerized ST (no vocalization). We found significant differences in HR and HRV parameters in both genders during ST when compared to relaxation (baseline) values. On the other hand, there were no significant differences in HR and HRV between males and females (except for a higher baseline HR in women). Also, reactivity to ST stress (difference between baseline and ST value) showed no gender differences. In conclusion, when performed in a supine position and without vocalization, the ST induces significant stress-related changes of HR and HRV in both genders, with no gender differences in the magnitude of reaction to ST stress. This experimental procedure can be used for correct examination of mental stress-related changes in the autonomic nervous system, and is particularly useful for examining mixed-gender experimental groups.