BACKGROUND: Despite the widely acknowledged benefit of exercise for patients with cancer, little evidence on the optimal timing of exercise on adverse effects of cancer treatment is available. OBJECTIVES: The aim of this study was to determine whether an exercise intervention initiated during chemotherapy is superior to an intervention initiated after chemotherapy for improving long-term cardiorespiratory fitness (peak oxygen uptake [VO]). METHODS: In this prospective, randomized clinical trial, patients scheduled to receive curative chemotherapy were randomized to a 24-week exercise intervention, initiated either during chemotherapy (group A) or afterward (group B). The primary endpoint was VO 1 year postintervention. Secondary endpoints were VO postintervention, muscle strength, health-related quality of life (HRQoL), fatigue, physical activity, and self-efficacy. Between-group differences were calculated using intention-to-treat linear mixed-models analyses. RESULTS: A total of 266 patients with breast (n = 139), testicular (n = 95), and colon cancer (n = 30) as well as lymphoma (n = 2) were included. VO immediately postintervention and 1 year postintervention did not differ between the 2 groups. Immediately postchemotherapy, patients in group A exhibited significantly lower decreases in VO (3.1 mL/kg/min; 95% CI: 2.2-4.0 mL/kg/min), HRQoL, and muscle strength and reported less fatigue and more physical activity than those in group B. CONCLUSIONS: Exercise can be safely performed during chemotherapy and prevents fatigue and decreases in VO, muscle strength, and HRQoL, in addition to hastening the return of function after chemotherapy. Also, if exercise cannot be performed during chemotherapy, a program afterward can enable patients to regain the same level of function, measured 1 year after completion of the intervention. (Optimal Timing of Physical Activity in Cancer Treatment [ACT]; NCT01642680).