Objective: Previous studies have found that an acute bout of exercise enhances cognitive performance in young adults and this relationship is mediated by increases in brain derived neurotrophic factor (BDNF). The purpose of the current study was to assess if resting BDNF may play a similar role in driving the relationship between overall cardiovascular fitness and executive function in midlife. Method: One hundred and sixty three participants, aged 40–60, underwent a general health assessment including a fasting blood draw to assess BDNF levels and a maximal oxygen uptake assessment (VO2 Max) to measure cardiovascular fitness. Participants also completed tests of global intelligence, executive functioning, and verbal memory. The executive function domain included Digit Span, the Stroop Word Color Test, and Trails B. A domain z score was calculated using sample-based z scores with timed test scores being inverted, so higher scores indicate better performance. Mediation was assessed using bootstrapping. Results: Higher VO2 max was related to better executive function performance, after controlling for age, education, baseline intelligence body mass index, systolic blood pressure, total cholesterol, and fasting glucose (VO2 max beta = 0.18, p = 0.014). However, BDNF did not appear to mediate this relationship (95% CI −0.0028 to 0.0134). Conclusion: Contrary to our hypothesis, plasma levels of BDNF did not mediate the relationship between higher cardiovascular fitness and better executive function performance in midlife suggesting that other mechanisms such as better insulin sensitivity may be responsible.