We evaluated 24-h time-domain heart rate variability (HRV) in 103 (46 females) healthy children and adolescents. Subjects were divided into four male and four female groups (ages 1-5, 6-10, 11-15, 16-20 years) and 24-h ambulatory Holter monitoring was performed. HRV was assessed by SDNN, SDNN index (SDNN-i), SDANN, rMSSD, pNN50. Males showed SDNN and SDANN values significantly higher than females while for SDNN-i, rMSSD, pNN50 there were no significant differences between sexes. With increasing age, there is a progressive and significant decrease of HR and increase of SDANN. On the other hand, SDNN, SDNNi, pNN50 and rMSSD increased significantly only between the first two age-groups. rMSSD and pNN50 were significantly related to body mass index. Thus, SDNN and SDANN, overall HRV measures, increased with age and were gender-related. HRV indices of parasympathetic function (rMSSD, pNN50) and SDNN-i increased up to 10 years of age and were gender-unrelated. These data demonstrate that in healthy children and adolescents there is a progressive modification of HRV that may reflect a progressive evolution of the autonomic nervous system, with different pattern measure-dependent. This paper enables us to compare, in future works, HRV in pediatric subjects in different groups according to the different HRV measures under examination.