Inflammation has been found to play a role in the etiology of cardiovascular disease as well as provoke endothelial dysfunction. Inflammatory cytokines associated with endothelial function are interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). IL-6 is exercise intensity dependent and has been shown to inhibit TNF-α expression directly. The aim of this study was to investigate the interaction of IL-6 and TNF-α on endothelial function in response to acute exercise in overweight men exhibiting different physical activity profiles.Methods and Procedures: Using a randomized mixed factorial design, 16 overweight men (8 active, maximal exercise capacity (VO2peak) = 34.2 ± 1.7, BMI = 27.4 ± 0.7 and 8 inactive, VO2peak = 30.9 ± 1.2, BMI = 29.3 ± 1.0) performed three different intensity acute exercise treatments. Brachial artery flow-mediated dilation (FMD) and subsequent blood samples were taken pre-exercise and 1 h following the cessation of exercise.Results: Independent of exercise intensity, the active group displayed a 24% increase (P = 0.034) in FMD following acute exercise compared to a 32% decrease (P = 0.010) in the inactive group. Elevated (P <0.001) concentrations of IL-6 following moderate (50% VO2) and high (75% VO2) intensity acute exercise were observed in both groups; however, concentrations of TNF-α were unchanged in response to acute exercise (P = 0.584).Discussion: The FMD response to acute exercise is enhanced in active men who are overweight, whereas inactive men who are overweight exhibit an attenuated response. The interaction of IL-6 and TNF-α did not provide insight into the physiological mechanisms associated with the disparity of FMD observed between groups.