The purpose of this study was to assess the effect of physical activity (PA) changes, measured by accelerometry, on telomere length (TL) in pediatric patients with abdominal obesity after a lifestyle intervention. One hundred and twenty-one children (7–16 years old) with abdominal obesity were randomized to the intervention (a moderately hypocaloric Mediterranean diet) or the usual care group (standard pediatric recommendations) for 22 months (a 2 month intensive phase and a subsequent 20 month follow-up). Both groups were encouraged to accumulate an extra 200 min/week of PA. TL was measured by MMqPCR. Data were analyzed in 102 subjects after 2 months and 64 subjects at the first 10 months of follow-up. Light PA level decreased in both groups after 12 months of intervention. At month 2, moderate to vigorous PA (MVPA) increased in the intervention group (+5.4 min/day, p = 0.035) and so did sedentary time in the usual care group (+49.7 min/day, p = 0.010). TL changes were positively associated (p < 0.050) with metabolic equivalents (METs), MVPA level, and number of steps, and were inversely associated with sedentary and light PA levels in the intervention group after the intensive phase. In conclusion, favourable changes in PA levels in the intensive phase of a lifestyle intervention could contribute to TL maintenance in a pediatric population with abdominal obesity. Novelty Changes in physical activity levels had a direct effect on telomere length, a biomarker of cellular aging and oxidative stress. PA advice based on The American College of Sports Medicine included in this intervention is easy to implement in primary care.