Background: Nearly all schools in the United States experienced shutdown followed by phased reopening during the COVID-19 pandemic, thereby limiting students’ opportunities for physical activity (PA). This study aimed to examine adolescents’ PA at school (PAS) and PA out-of-school (PAO), screen-based sedentary behaviors (SbSB), health-related fitness, and knowledge understanding about PA and fitness before and during the pandemic. Methods: Three rounds of data were collected: Time 1 pre-pandemic (January 2020; n = 405), Time 2 schools partially reopened (February 2021; n = 412), and Time 3 schools fully reopened (March 2021; n = 450). Adolescents completed the Youth Activity Profile, the 20 m Progressive Aerobic Cardiovascular Endurance Run (PACER) test and Plank test, and a written test, to measure the behaviors (T1, T2, T3), fitness (T2–T3), and knowledge (T1, T2, T3), respectively. Results: Inferential statistical analyses revealed a significant time effect for the behaviors and fitness. From T1 to T2 PAO decreased but PAS increased; whereas SbSB decreased at T3 compared to T1 and T2. Health-related fitness improved from T2 to T3. Further, the change patterns for SbSB varied by grade, and those for knowledge understanding varied by gender. Conclusion: The findings confirm the disruptive impact of the COVID-19 pandemic on adolescents’ active living but varied by school grade and gender. The favorable changes from T2 to T3 observed for fitness and other constructs may be partially attributable to an interrupted fitness education intervention. The findings may guide the design and evaluation of future interventions addressing the physical inactivity pandemic during public health crises (e.g., COVID-19).