Previous research analyzing the differences in physical fitness and body composition between active and sedentary adolescents aged 12–16 has not provided conclusive results. For this reason, a systematic review with meta-analysis was conducted to provide an overview of the results obtained to date. The objectives of this systematic review and meta-analysis were to investigate the differences in the physical fitness and body composition of adolescents who engaged in daily physical activity and those who were inactive. A search in PubMed, EBSCO, and Web of Sciences databases was performed. A total of 13,884 articles were reviewed and 11 were included in the meta-analysis. In the physical fitness performance, significantly higher values in cardiorespiratory fitness, hamstring and lower back flexibility, sit-ups and upper limb resistance were found in active compared to the inactive participants. In body composition, the inactive group showed significantly higher values in variables related to body fat, mainly in body fat percentage, fat mass and fat mass index compared to the active group. The results revealed that maintaining an active lifestyle through physical activity is a determining factor in improving the physical fitness and body composition of adolescents aged 12–16 years. The study design of the systematic review was previously registered in PROSPERO with code CRD42021241975. https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=241975.