In the Amsterdam Growth and Health Longitudinal Study (AGAHLS), a group of apparently healthy males and females (n = 200) were interviewed about their physical activities on eight separate occasions over a period of 20 years between 13 and 33 years of age (multi-measured group: MM). Information about their health was given based on their personally measured lifestyle (activity, diet, smoking) and biological risk characteristics for chronic diseases (medical check-ups). A comparable group of boys and girls (n = 200) was only measured on two occasions (bi-measured group: BM): at 13 and 33 years. Physical activity was estimated with a structured interview. Total physical activity and sports activity were estimated in three intensity levels (light, moderate, and heavy). It was hypothesized that the eight repeated medical check-ups with health information in the MM group would result in a healthier lifestyle with respect to the determinants and levels of habitual physical activity compared to the BM group. Contrary to the hypothesis, males and females in the BM group showed a significantly higher increase or a lower decrease in physical activities compared to the MM group. This negative effect on the physical activity pattern at 33 years in the MM group may have been caused by more underreporting of physical activities than in the BM group. In conclusion, there does not appear to be a significant effect of long-term (multi-measured) health information with medical check-ups during adolescence and young adulthood on level of physical activity in males and females at 33 years of age.