Background: This study examined the effects of a five-month lockdown due to COVID-19 pandemic on physical fitness parameters in urban adolescent male and female students. Methods: Two hundred and ninety-three male and female students (age: 15.8 ± 0.3 years) who attended the fourth grade of the same high school during the years 2016–2017 (first control group), 2018–2019 (second control group) and 2020–2021 (lockdown group) took part in the present study. Results: The percentage of overweight and obese students, according to body mass index, increased in males from 16.0% (2016–2017) and 14.6% (2018–2019), to 36.7% in 2020–2021 (p < 0.01), and in females from 8.6% (2016–2017) and 7.0% (2016–2017), to 25.6% in 2020–2021 (p < 0.01). Lower body fitness, as assessed by jumping, sprinting and agility tests, was impaired for both males and females after the lockdown compared with the 2016–2017 and 2018–2019 cohorts (vertical jumps: 10.4–15.1%; p < 0.01; d = 0.58–1.01, 30 m sprint: 3.7–4.9%; p < 0.01; d = 0.62–0.74; 505 agility test: from 6.1% to 9.4%; p < 0.01; d = 0.80–1.04). However, flexibility and performance in upper-body fitness tests (handgrip maximum isometric strength and medicine ball throws with different loads) was significantly reduced only in males after the lockdown (p < 0.05 to 0.01). Conclusions: These results suggest that a five-month lockdown negative influenced the physical fitness of adolescent students. Notably, greater reductions were observed in upper body strength, power and flexibility in males than in females. These results highlight the need to maintain strength, power and body mass during long periods of inactivity in adolescent populations.