The dissociation of effects of age, time of assessment and cohort is a well-known challenge in developmental science. We examined effects of time of assessment in the school year on children’s physical fitness using data from 75,362 German third-graders from seven cohorts. Children were tested once either in the first or second school term of third grade. Tests examined cardiorespiratory endurance (6-min run), coordination (star-run), speed (20-m sprint), lower (standing long jump) and upper (ball-push test) limbs muscle power, and flexibility (stand-and-reach test). We estimated the effect of time of assessment using a regression discontinuity design specified in a linear mixed model with random factors child and school and adjusted for age, sex, and cohort effects. Coordination, speed, and upper limbs muscle power were better in second compared to first school term, with boys exhibiting a larger increase of upper limbs muscle power than girls. There was no evidence for changes in cardiorespiratory endurance, lower limbs muscle power, and flexibility between assessments. Previously reported age and sex effects as well as secular fitness trends were replicated. There is thus evidence for improvement of some physical fitness components beyond age and cohort effects that presumably reflects the benefit of physical activity in physical education and other settings. Effects of assessment time should be taken into consideration in performance-based grading or norm-based selection of children.