To identify the effects of an intervention in Physical Education classes and handball training on health-related physical fitness in adolescent girls. Methods: This is a quasi-experimental study with 26 girls (10 to 16 years old). Girls who reached 75% of participation in handball training (HT) (n= 12) composed the HT group. Girls who played only during Physical Education composed the PE group (n=14). Waist circumference (WC), body mass index (BMI), cardiorespiratory fitness (CRF), abdominal strength (ABDS), and flexibility were evaluated using PROESP-Br procedures. The delta of glass (DGlass) was adopted as the measure of the effectiveness of these analyses. Results: The girls involved in HT reduced WC and increased CRF, ABDS, and flexibility with large effect sizes (33% to 75% of individual improvements). We observed fewer improvements in the PE group, with a variation of 14% to 35% of positive effects. Conclusion: The HT in after-class time together Physical Education classes provide improvements until 75% in the physical fitness of adolescent girls. In another way, Physical Education classes also provided these effects; however, it was in a minor proportion of about 35%.