Purpose: This study examined differences in measures of health-related physical fitness in adolescents before and after extended school closures due to COVID-19. Method: The sample consisted of 298 students (135 males and 163 females) from a laboratory high school. Data were collected through FITNESSGRAM assessments. A repeated-measures multivariate analysis of covariance was calculated to analyze differences in fitness before and after COVID-19 closures, including McNemar-Bowker and McNemar tests. Results: Statistically significant differences were identified for Progressive Aerobic Capacity Endurance Run (-4.2%; 1.8 ml·kg[superscript -1]·min[superscript -1]) and curl-up (-12.5%; 7.9 repetitions). In addition, 18.8% fewer students were classified within the Healthy Fitness Zone for Progressive Aerobic Capacity Endurance Run, 4% for curl-up, 10.8% for push-up, and 6.4% for sit and reach. Conclusion: Results of this study demonstrate that there was a significant decline in physical fitness for secondary students during extended school closures and social isolation as a result of COVID-19.