Endurance exercise is an effective therapeutic intervention with substantial pro-healthspan effects. Male Drosophila respond to a ramped daily program of exercise by inducing conserved physiological responses similar to those seen in mice and humans. Female flies respond to an exercise stimulus but do not experience the adaptive training response seen in males. Here, we use female flies as a model to demonstrate that differences in exercise response are mediated by differences in neuronal activity. The activity of octopaminergic neurons is specifically required to induce the conserved cellular and physiological changes seen following endurance training. Furthermore, either intermittent, scheduled activation of octopaminergic neurons or octopamine feeding is able to fully substitute for exercise, conferring a suite of pro-healthspan benefits to sedentary Drosophila. These experiments indicate that octopamine is a critical mediator of adaptation to endurance exercise in Drosophila. Chronic exercise causes stereotypical adaptations in muscle and adipose tissue of Drosophila. Sujkowski et al. show that these adaptations require the activity of octopaminergic neurons. Differences in octopaminergic activity control sexual dimorphism in exercise response. Both octopamine feeding and stimulation of octopaminergic neurons can substitute for endurance exercise.