The purpose of this study was to test the hypothesis that physical activity (PA), measured over a period of 23 years, is beneficial to aerobic fitness (VO2max) in boys and girls (13-36 years) who were enrolled in the Amsterdam Growth and Health Longitudinal Study (AGAHLS). PA was measured using a standardized activity interview. VO2max was assessed directly with a maximal running test on a treadmill. To assess the longitudinal relationship between PA and VO2max, different longitudinal analyses were carried out over different age periods, correcting for various confounders such as lifestyle parameters, biological parameters, and initial VO2max. Highly significant relationships (p < .05) were observed between PA and VO2max in four of the five analyses. However, in an autoregression analysis, when current PA has been related to the future change in VO2max, the results are not any more significant (p > .05). Analysis of the data of PA and VO2max from the AGAHLS population does not fully support the hypothesis that PA affects VO2max.