Key autonomic functions are in continuous development during adolescence which can be assessed using the heart rate variability (HRV). However, the influence of different demographic and physiological factors on HRV indices has not been fully explored in adolescents. In this study we aimed to assess the effect of age, gender, and heart rate on HRV indices in two age groups of healthy adolescents (age ranges, 13–16 and 17–20 years) and two groups of healthy young adults (21–24 and 25–30 years). We addressed the issue using 5-min ECG recordings performed in the sitting position in 255 male and female participants. Time, frequency, and informational domains of HRV were calculated. Changes in HRV indices were assessed using a multiple linear regression model to adjust for the effects of heart rate, age, and gender. We found that heart rate produced more significant effects on HRV indices than age or gender. There was a progressive reduction in HRV with increasing age. Sympathetic influence increased with age and parasympathetic influence progressively decreased with age. The influence of gender was manifest only in younger adolescents and young adults. In conclusion, age, gender, and particularly heart rate have a substantial influence on HRV indices, which ought to be considered to avoid biases in the study of the autonomic nervous system development. The lack of the gender-related effects on HRV indices in late adolescence could be related to non-completely achieved maturity of the autonomic mechanisms, which deserves further exploration.