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To assess changes in health-related quality of life (HRQoL) in children and adolescents over a 3-year period and to examine factors associated with change.A representative sample of Spanish children and adolescents aged 8–18 years and their parents completed the KIDSCREEN-52 questionnaire at baseline and again after 3 years. Data were also collected on gender, pubertal development (PD), and family socio-economic status (SES). Change in HRQOL over time was evaluated using effect sizes (ES). Generalized estimating equations (GEE) were used to analyze associations among changes in KIDSCREEN scores, socio-demographic factors, and pubertal development.Response rate at follow-up was 54% (n = 454). Overall, HRQoL worsened in eight out of the ten KIDSCREEN dimensions, with ES ranging from −0.10 (Moods and Emotions) to −0.34 (Psychological Well-being). The decrease was most marked in the intermediate age group (13–17 years of age at follow-up) and in girls. In the GEE models, pubertal development more strongly impacted changes in girls than in boys.In this representative, population-based sample of children and adolescents, moderate decrements in HRQoL were observed after 3 years. Changes were particularly important among girls and in relation to pubertal development. These results could provide useful reference data for other longitudinal studies in population sub-groups.