This study examined how the degree of fitness affects several psychophysiological responses to stress in elite sportswomen and physically active women. Trait anxiety and mood were evaluated, and salivary testosterone (Tsal) and cortisol (Csal) concentrations determined before and after a maximal ergometry. Afterwards, subjects carried out the Stroop task, where heart rate (HR) and skin conductance level (SCL) were continuously recorded. At baseline, elite sportswomen had worse mood, and lower cardiovascular basal measures and testosterone:cortisol ratio. In the Stroop task, they showed higher SCL reactivity and worse recovery with respect to the baseline values than physically active women. There was a positive relationship between Tsal and HR as well as between negative mood and SCL, and a negative one between negative mood and HR. In women, the degree of fitness moderates he electrodermal responses to acute stress, a specific pattern of relationship among different psychophysiological variables being found.