Cross-sectional studies suggest that moderate physical activity is associated with enhanced resting immune function; however, few randomized controlled trials have investigated this link. We investigated the effect of 12-months aerobic exercise, relative to stretching control, on in vitro immune function in a randomized controlled trial of 115 postmenopausal, overweight or obese, sedentary women, aged 50-75 years. The exercise goal was 45 min.d-1, 5 d.wk-1. Control women participated in 1 d.wk-1 stretching classes. Immune markers (natural killer (NK) cell cytotoxicity, T lymphocyte proliferation, immune cell counts and phenotypes, and serum immunoglobulins) were assessed at baseline, 3-months, and 12-months, under strict blood-draw criteria. General estimation equations evaluated intervention effects at three and twelve months, controlling for baseline. Of the 115 women who began the trial, blood samples were available from 109 at 3-months (95%) and 108 at 12-months (94%). From baseline to 12-months, the exercise group participated in 87% of the prescribed physical activity minutes per week and increased [latin capital V with dot above]O2max by 13.8%: controls experienced no change in fitness. The main outcomes, NK cell cytotoxicity and T lymphocyte proliferation, did not differ between groups at 3-months and at 12-months. Secondary outcome and subgroup (e.g. stratification by baseline categories of BMI, immune status, CRP, and age) analyses did not show any clear patterns of association. This 12-month randomized controlled trial showed no effect of aerobic exercise on in vitro immune function, despite excellent retention, high adherence, and demonstrable efficacy of the exercise intervention.