The present study aimed to identify the impact of social distancing measures on adolescents’ physical activity (PA) levels and well-being during the implementation of COVID-19 restrictive measures. There were 438 participants (207 boys and 231 girls), aged 12 to 15 years old (M= 13.5,SD= 0.55). They completed online questionnaires on well-being and PA in three waves (December 2020, February 2021, and June 2021). Correlation analyses were conducted to examine the relationship between well-being and PA variables in the three measurements. Additionally, separate three-way repeated-measures ANOVAs were conducted to capture possible differences in students’ moderate to vigorous physical activity (MVPA) levels, life satisfaction, and subjective vitality among the three measurements due to gender, age, and interaction between gender and age. A significant relation emerged between the MVPA variables and well-being. In all measurements, adolescents’ PA levels did not meet the World Health Organization (WHO) recommendations of at least 60 min per day in MVPA. Students’ MVPA levels, life satisfaction, and subjective vitality were significantly higher in the third measurement compared to the first and second ones. Moreover, significant differences emerged in life satisfaction and subjective vitality between boys and girls in the first and third measurements, respectively. The COVID-19 restrictions appeared to negatively influence adolescents’ PA and well-being. Policymakers aimed at facilitating adolescents’ well-being in a similar situation in the future should not adopt measures restricting the participation of adolescents in PA.