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This study aimed to determine whether higher levels of physical activity (PA) and less sedentary behavior (SB) are associated with less inflammation, indicated by inflammatory and hemostatic biomarkers, in older men.A cross-sectional study of 1139 men, from the British Regional Heart Study (mean ± SD age = 78 ± 5 yr), and longitudinal analyses of 490 men with two PA measures 1 yr apart were used in this study. Single fasting venous blood samples were analyzed for several biomarkers. PA and SB were measured using ActiGraph GT3X accelerometers. Total time and time spent in bouts of moderate to vigorous PA (MVPA), light PA, and SB were derived. Linear regression analyses were used to investigate associations.Cross-sectionally, higher total PA, daily steps, and MVPA were all associated with lower levels of interleukin 6 (IL-6), C-reactive protein (CRP), tissue plasminogen activator (tPA), von Willebrand factor (vWF), and D-dimer, whereas higher levels of SB were associated with higher levels of IL-6, CRP, and tPA. Each additional 10 min of MVPA was associated with a 3.2% lower IL-6 (95% confidence interval [CI] = −4.5% to −1.8%), 5.6% lower CRP (95% CI = −7.8 to −3.3), 2.2% lower tPA (95% CI = −3.0 to −1.4), 1.2% lower vWF (95% CI = −2.1 to −0.3), and 1.8% lower D-dimer (95% CI = −2.9 to −0.7), and for CRP, vWF, and D-dimer independently of SB. Associations between SB and IL-6 or tPA were independent of MVPA. Longer bouts of PA or SB were not more strongly associated with outcomes than shorter bouts. Longitudinal analyses were inconsistent with these findings, possibly because of power limitations.Although PA (particularly MVPA) was generally associated with inflammatory and hemostatic biomarkers, we found no evidence that longer bouts were more important than shorter bouts.