Valid assessments of physical activity (PA) and cardiorespiratory fitness (CRF) is essential in epidemiological studies to define dose-response relationship for e.g. formulating thorough recommendations of an appropriate pattern of PA to maintain good health. The aim of this study was to validate the Danish step test, the physical activity questionnaire Active-Q and self-rated fitness against directly measured maximal oxygen uptake (VO2 max). A population based subsample (n=125) was included from the 'Diet, Cancer and Health - Next Generations' (DCH-NG) cohort which is under establishment. Validity coefficients, which express the correlation between measured and 'true' exposure, were calculated and misclassification across categories was evaluated. The validity of the Danish step test was moderate (women: r=0.66, and men: r=0.56), however, men were systematically underestimated (43% misclassification). When validating the questionnaire-derived measures of PA, leisure time physical activity was not correlated with VO2 max. Positive correlations were found for sports overall, but these were only significant for men: total hours per week of sports (r=0.26), MET-hours per week of sports (r=0.28) and vigorous sports (0.28) alone were positively correlated with VO2 max. Finally, the percentage of misclassification was low for self-rated fitness (women: 9% and men: 13%). Thus, self-rated fitness was found to be a superior method to the Danish step test, as well as being less cost prohibitive and more practical than the VO2 max method. Finally, even if correlations were low, they support the potential for questionnaire outcomes, particularly sports, vigorous sports and self-rated fitness to be used to estimate CRF. This article is protected by copyright. All rights reserved.