Objective: To assess whether breath acetone concentration can be used to monitor the effects of a prolonged physical activity on whole body lipolysis and hepatic ketogenesis in field conditions. Methods: Twenty-three non-diabetic, 11 type 1 diabetic, and 17 type 2 diabetic subjects provided breath and blood samples for this study. Samples were collected during the International Four Days Marches, in the Netherlands. For each participant, breath acetone concentration was measured using proton transfer reaction ion trap mass spectrometry, before and after a 30-50 km walk on four consecutive days. Blood non-esterified free fatty acid (NEFA), beta-hydroxybutyrate (BOHB), and glucose concentrations were measured after walking. Results: Breath acetone concentration was significantly higher after than before walking, and was positively correlated with blood NEFA and BOHB concentrations. The effect of walking on breath acetone concentration was repeatedly observed on all four consecutive days. Breath acetone concentrations were higher in type 1 diabetic subjects and lower in type 2 diabetic subjects than in control subjects. Conclusions: Breath acetone can be used to monitor hepatic ketogenesis during walking under field conditions. It may, therefore, provide real-time information on fat burning, which may be of use for monitoring the lifestyle interventions.Obesity (2014) 22, 980-983.