The physiological roles of isoprene, which is one of the many endogenous volatile organic compounds contained in exhaled breath, are not well understood. In recent years, exhaled isoprene has been associated with the skeletal muscle. Some studies have suggested that the skeletal muscle produces and/or stores some of the isoprene. However, the evidence supporting this association remains sparse and inconclusive. Furthermore, aging may affect breath isoprene response because of changes in the skeletal muscle quantity and quality. Therefore, we investigated the association between the breath isoprene excretion ([Formula: see text]) and skeletal muscle mass in young (n = 7) and old (n = 7) adults. The participants performed an 18 min cycling exercise after a 3 min rest. The workload corresponded to an intensity of 30% of the heart rate reserve, as calculated by the Karvonen formula. The exhaled breath of each participant was collected during the exercise test. We calculated [Formula: see text] from the product minute ventilation and isoprene concentration and, then, investigated the relationships between [Formula: see text] and muscle mass, which was measured by multi-frequency bioelectrical impedance analysis. Importantly, muscle mass persisted as a significant determinant that explained the variance in [Formula: see text] at rest even after adjusting for age. Furthermore, the muscle mass was a significant determinative factor for [Formula: see text] response during exercise, regardless of age. These data indicated that skeletal muscle mass could be one of the determinative factors for [Formula: see text] during rest and response to exercise. Thus, we suggest that the skeletal muscle may play an important role in generating and/or storing some of the endogenous isoprene. This new knowledge will help to better understand the physiological functions of isoprene in humans (Approval No. 20190079).