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Short leukocyte telomere length (LTL) has become a hallmark characteristic of aging and is associated with higher morbidity and mortality. Physical activity (PA) has been implicated in attenuating age-induced diseases by, for example, preserving LTL. Results from studies of the relationship between PA and LTL have been mixed, which might be because PA was assessed over a short period of time. There have been few studies in which investigators have examined the association between LTL and cardiorespiratory fitness (CRF), an enduring trait influenced by chronic habituation of PA that takes place over months or years. The purpose of the present study was to examine the association between CRF and LTL in a national sample of US adults who were 20-49 years of age from the 1999-2002 National Health and Nutrition Examination Survey (n = 1,764). LTL was assessed using quantitative polymerase chain reaction, and CRF assessed using a treadmill-based exercise test. After adjustments, compared with subjects in the lowest CRF tertile, those in the middle tertile (β = 0.03, 95% confidence interval: 0.005, 0.06; P = 0.02) and upper tertile (β = 0.05, 95% confidence interval: 0.004, 0.09; P = 0.04) had longer LTL. These findings suggest that higher CRF is associated with longer LTL.