It was established that the COVID-19 pandemic resulted in decreased physical activity levels, potentially leading to reduced physical fitness. The aim of this research was to investigate the impact of the COVID-19 lockdown on fitness indices in adolescents. We observed high school adolescents (33% girls; aged 15-17 years) divided into two cohorts. The first cohort (control; n = 48) included adolescents who were in school during the 2018/2019 year, while the second cohort (lockdown; n = 66) was observed during the school year 2019/2020 when COVID-19 lockdown measures were imposed. Variables included body height, body mass, BMI, sit-ups, and the 600 m dash. Both cohorts were tested at the beginning and at the end of the observed school years. A factorial analysis of variance for repeated measurements evidenced an improvement in sit-ups and the 600 m dash in the control, and a decrease of the same capacities in the lockdown cohort. In the lockdown cohort, a decrease in muscular fitness was more evident in boys than in girls. No differential changes between cohorts were evidenced in anthropometrics. Results suggest that the COVID-19 lockdown negatively influenced muscular fitness status in adolescents, especially in boys.