Physical fitness represents an essential component of military readiness and a key prerequisite for the successful completion of military training and future service. The aim of this study was to analyse and compare the annual physical fitness assessment results among University of Defence students in the Czech Republic from 2022 to 2025, with a focus on differences between faculties. The analysis was based on administrative data from standardized testing and combined both cross-sectional and longitudinal approaches. Endurance was assessed using the 12-minute run (Cooper test), while the strength component was evaluated by the maximum number of pull-ups for males and the flexed-arm hang for females. The results showed no statistically significant differences between faculties in endurance performance among either males or females. Statistically significant differences between faculties were found only among males in the strength discipline, with the highest values observed among students of the Military Faculty of Medicine. The longitudinal analysis revealed that physical performance development during the study is not linear and varies by sex, faculty, and discipline type. For males, a clear increase in strength performance was observed across all faculties, while endurance performance showed a less stable pattern, including a temporary decline in some groups. The transition between the first and second year of study proved to be a critical period, during which some students experienced a decrease in endurance performance alongside an improvement in strength abilities. The findings of this study may contribute to the optimization of physical training during military university education and to a better consideration of the specific characteristics of individual study programmes.