DHEA is marketed and readily available as a daily nutritional supplement to counteract the effects of aging. The effect of DHEA administration on 24-hour plasma cortisol profiles has not been investigated. In this single-blind placebo-controlled crossover study, the effect of DHEA administration on cortisol concentrations was evaluated in healthy older women and men. Once each morning, subjects took either placebo (Days 1 to 7, and 23 to 29) or oral DHEA 200 mg (Days 8 to 22: doses 1 to 15). Twenty-four hour DHEA and cortisol concentrations were measured on Day 1 (placebo), Day 8 (DHEA dose 1), Day 15 (DHEA dose 8), Day 22 (DHEA dose 15), and Day 29 (placebo washout dose 7). DHEA administration resulted in a decrease in plasma cortisol concentrations (mean, peak, and/or AUC) in healthy older women and men. The cortisol-lowering effect of DHEA was more pronounced in women than in men in our study; pairwise differences in concentrations between days showed that relative to Day 1, cortisol was lower on Days 15, 22, and 29 in women (p = 0.0001) and on Day 15 in men (p = 0.002). The mechanism by which DHEA lowers plasma cortisol concentrations merits further investigation.