Acetone, which is exhaled with breath, is a by-product of lipolysis and could be used as a simple, useful indicator of lipolysis in the body because, unlike blood sampling, it can be measured non-invasively and repeatedly. Breath acetone concentration, however, is known to be affected by several factors such as exercise and food. We designed the experiments to evaluate the mixed effect on breath acetone of exercise and food ingestion in order to enhance the usefulness of breath acetone for monitoring fat loss. Seven healthy males performed moderate exercise for twice of 45 min with an interval of 15 min then rested for 4 h. Exhaled air was sampled every 15 min throughout the experiment. The subjects took one of four types, sugar-rich, balanced, protein-rich and fat-rich, of food for lunch one hour after the exercises or kept fasting. In the case of fasting, breath acetone kept increasing significantly (p < 0.05) compared with the rest value after the exercises until the end of the experiment. In contrast, in the case of taking any type of food, the change in breath acetone varied according to the food type. In the case of taking sugar-rich food, breath acetone significantly decreased (p < 0.05) compared with the fasting case. This decrease might be due to a suppression of acetone production when carbohydrates such as sugar are supplied to a body in the fasting condition. In contrast, in the case of taking fat-rich food, breath acetone showed the higher level than the fasting case. This additional increase might be attributable to the promotion of ketone bodies production, including acetone, due to the ingestion of medium chain triglycerides contained in the fat-rich food. We should therefore consider exercise and food ingestion in using breath acetone as a non-invasive indicator of lipolysis.