Naturally 13C-enriched carbohydrate has been used to label the liver glycogen pool for metabolic studies. The utilization of this glycogen was then monitored by the appearance of 13CO2 in breath. Using this method, it is assumed that during sedentary fasting the contribution of muscle glycogen towards oxidation is negligible. We investigated the influence of a different level of 13C enrichment of muscle glycogen on the 13C enrichment of breath CO2 while the breath test was carried out. In six healthy volunteers, the muscle glycogen stores were grossly depleted by a cycling exercise prior to consumption of the 13C-enriched diet which was given over a 10 h period. The oxidation of liver glycogen was measured during an 18 h sedentary fast. The results were compared with a control group who had not depleted their muscle glycogen before labelling. A higher 13C enrichment of muscle glycogen did not interfere with two parameters of liver glycogen oxidation, i.e. the duration of the plateau phase of 13CO2 and the return to baseline time. It was also shown that the 13C-labelled muscle glycogen was still available after the 18 h fast because a strenuous exercise led to a rapid 13CO2 enrichment. It is concluded that muscle glycogen 13C enrichment does not invalidate a 13CO2 breath test to measure liver glycogen oxidation during a sedentary fast.