Background—The association of physical activity (PA), measured three ways, and biomarkers were compared in a sample of adolescents. Methods—PA data were collected on two cohorts of adolescents (N=700) in the Twin Cities, Minnesota, 2007–2008. PA was measured using two survey questions (Modified Activity Questionnaire (MAQ)), the 3-Day Physical Activity Recall (3DPAR), and accelerometers. Biomarkers included systolic (SBP) and diastolic blood pressure (DBP), lipids, percent body fat (%BF) and body mass index (BMI) percentile. Bivariate relationships among PA measures and biomarkers were examined followed by generalized estimating equations for multivariate analysis. Results—The three measures were significantly correlated with each other (r=0.22–0.36, P<0.001). Controlling for study, puberty, age and gender, all three PA measures were associated with %BF (MAQ=-1.93, P<0.001; 3DPAR=-1.64, P <0.001; accelerometer=-1.06, P =0.001). The MAQ and accelerometers were negatively associated with BMI percentile. None of the three PA measures were significantly associated with SBP or lipids. The percentage of adolescents meeting the national PA recommendations varied by instrument. Conclusions—All three instruments demonstrated consistent findings when estimating associations with %BF, but were different for prevalence estimates. Researchers must carefully consider the intended use of PA data when choosing a measurement instrument.