Abstract Background Adolescents with overweight and poor physical fitness have an increased likelihood of developing cardiovascular diseases during adulthood. In Ecuador, a health promotion program improved the muscular strength and speed-agility, and reduced the decline of the moderate-to-vigorous physical activity of adolescents after 28 months. We performed a sub-group analysis to assess the differential effect of this intervention in overweight and low-fit adolescents. Methods We performed a cluster-randomized pair matched trial in schools located in Cuenca–Ecuador. In total 20 schools (clusters) were pair matched, and 1440 adolescents of grade 8 and 9 (mean age of 12.3 and 13.3 years respectively) participated in the trial. For the purposes of the subgroup analysis, the adolescents were classified into groups according to their weight status (body mass index) and aerobic capacity (scores in the 20 m shuttle run and FITNESSGRAM standards) at baseline. Primary outcomes included physical fitness (vertical jump, speed shuttle run) and physical activity (proportion of students achieving over 60 min of moderate–to-vigorous physical activity/day). For these primary outcomes, we stratified analysis by weight (underweight, normal BMI and overweight/obese) and fitness (fit and low fitness) groups. Mixed linear regression models were used to assess the intervention effect. Results The prevalence of overweight/obesity, underweight and poor physical fitness was 20.3 %, 5.8 % and 84.8 % respectively. A higher intervention effect was observed for speed shuttle run in overweight (β = −1.85 s, P = 0.04) adolescents compared to underweight (β = −1.66 s, P = 0.5) or normal weight (β = −0.35 s, P = 0.6) peers. The intervention effect on vertical jump was higher in adolescents with poor physical fitness (β = 3.71 cm, P = 0.005) compared to their fit peers (β = 1.28 cm, P = 0.4). The proportion of students achieving over 60 min of moderate-to-vigorous physical activity/day was not significantly different according to weight or fitness status. Conclusion Comprehensive school-based interventions that aim to improve diet and physical activity could improve speed and strength aspects of physical fitness in low-fit and overweight/obese adolescents. Trial registration Clinicaltrials.gov identifier NCT01004367 . Registered October 28, 2009.