Sex-based differences in inflammatory responses to exercise may be mediated by estrogen through increased muscle membrane stability and/or inhibited cytokine production. In this study, in vivo effects of estrogen on systemic inflammation-related responses to exercise were assessed in healthy men. In a double-blind, placebo-controlled, crossover design, 11 men cycled for 90 min at 65% V̇o2 max after 8 days of 17β-estradiol supplementation (ES; 2 mg/day) or placebo (PL; glucose polymer). After a 2-wk washout, exercise was repeated after 8 days on the alternate treatment. Blood was collected pre- and postexercise to determine IL-6, soluble intercellular adhesion molecule-1 (sICAM-1), neutrophil counts, and cortisol. Preexercise serum was assayed for sex hormones. ES increased estradiol (133 ± 71 to 840 ± 633 pmol/l, P = 0.005) and reduced testosterone (19.9 ± 3.7 to 16.1 ± 3.9 nmol/l, P = 0.007). Exercise increased cortisol ( P = 0.02), IL-6 ( P < 0.001) and neutrophil counts ( P < 0.001) with no influence on sICAM-1 ( P = 0.34) and no effect of ES on these changes. Postexercise IL-6 and neutrophil counts were correlated ( r = 0.58, P = 0.005); postexercise IL-6 and cortisol ( r = 0.18, P = 0.43) and postexercise cortisol and neutrophil counts ( r = 0.06, P = 0.78) were not. Postexercise sICAM-1 was not correlated with the above variables ( P ≥ 0.79). In conclusion, 8 days of ES in healthy men did not influence systemic inflammation-related responses to acute exercise. Future studies should investigate 17β-estradiol effects on IL-6 production and neutrophil infiltration within skeletal muscle during and after exercise.