The association of physical activity (PA) with the prevention and treatment of various diseases is well known. The issue of insufficient physical activity among university students has worsened due to the effects of the COVID-19 pandemic. The aim of this research is to examine the social, lifestyle, and health factors associated with moderate and high levels of PA in two cross-sectional studies conducted before and after COVID-19 involving 1,266 medical students from five universities in Serbia. Two cross-sectional studies were conducted on the population of fifth-year medical students from five different universities in Serbia before and after the COVID-19 pandemic (in 2019 and 2023). The research instrument was a questionnaire specially designed based on similar research. The mean energy expenditure in MET-minutes per week increased from 2,265.26 ± 1,930.67 in 2019 to 2,871.65 ± 2,301.99 in 2023 (p <
0.001). Multivariate logistic regression analysis with high PA as an outcome variable for the entire sample showed the association of high PA with the University of Kosovska Mitrovica and score on the Zung anxiety scale. Multivariate logistic regression analysis with high PA as an outcome variable for 2019 showed the association of high PA with the University of Kosovska Mitrovica, number of meals per day, and score on the Zung anxiety scale. Multivariate logistic regression analysis with high PA as an outcome variable for 2023 showed the association of high PA with the University of Kosovska Mitrovica, number of meals per day, and score on the Zung anxiety scale. The average energy expenditure was significantly higher in 2023. Factors such as studying in Kosovska Mitrovica, scores on the Zung Anxiety Scale, alcohol consumption, binge drinking in the past month, and the number of meals consumed per day were associated with either moderate or high levels of PA or both.