Background: Thyroid hormones are important metabolic regulators exerting effects in multiple systemic functions including muscular and cardiorespiratory function. Thyroid hormones may influence physical activity levels. However, there are currently no studies evaluating the association between thyroid function and physical activity levels in the general population. Methods: In a population-based cohort study between 2006 and 2013, we assessed the cross-sectional and longitudinal (with a mean follow-up time of 5 years) association of serum thyrotropin (TSH) and free thyroxine (fT4) with physical activity (metabolic equivalent task [MET] hours per week). Information on physical activity was collected using a validated questionnaire (Longitudinal Aging Study Amsterdam, median 22.50 MET hours per week). The association of TSH and fT4 with physical activity was examined using linear regression models in the cross-sectional and longitudinal analyses, adjusted for age, sex, lifestyle factors, and cardiovascular disease. In sensitivity analyses, we examined the association between thyroid function and physical activity including only participants within the reference range of thyroid function. We additionally examined moderate and vigorous physical activity separately as outcomes. Results: We included 2470 participants for the cross-sectional analysis (mean age 57.3 years, 58% women) and 1907 participants for the longitudinal analysis (mean age 56.9 years). There was no association between TSH (mIU/L) or fT4 (ng/dL) and physical activity (b = 0.65, 95% confidence interval [CI, -1.67 to 2.98] and b = 2.76, [CI -7.15 to 12.66], respectively) on cross-sectional analysis. Similarly, in the longitudinal analyses, we observed no association of TSH (b = 1.16, [CI -1.31 to 3.63]) or fT4 (b = -6.63, [CI -17.06 to 3.80]) with physical activity. Conclusions: We did not observe an association between the endogenous thyroid hormone level and total physical activity. Further studies need to be performed to evaluate whether thyroid hormone replacement therapy is associated with physical activity.