Regular endurance training increases fat oxidation during exercise at a given exercise load and decreases carbohydrate utilisation. In addition to training status the daily dietary macronutrient intake as well as the consumption of carbohydrate containing beverages and/or food during exercise will influence fuel utilisation during exercise. When ultra-endurance exercise including several different exercise modalities is performed more or less continuously for several days fat oxidation during exercise is increased both at rest and during submaximal exercise (1). However, the increased fat oxidation may well be due to decreased glycogen stores and inability to maintain energy balance, both of which will increase fat utilization during exercise. To study the effect of excessive prolonged exercise on maximal fat oxidation during exercise 14 days of very prolonged exercise was performed by 6 elderly male subjects. The study adhered to the Declaration of Helsinki and was approved by the Science Ethical Committee of the Copenhagen Region (H3-2011-008) and also registered at clinicaltrials.gov (NCT02353624). Over the 14 days the subjects performed approximately 10½ hours of cycle exercise per day. During the 14 days the subjects consumed an ad lib high carbohydrate diet and before and approx. 30-34 hours after completion of the last of the 14 exercise days maximal fat oxidation as well as maximal oxygen uptake was measured during a graded cycle exercise protocol in the overnight fasted condition. Before and after the 14 days muscle biopsies from vastus lateralis and a blood sample under resting conditions were obtained. Interestingly a marked decrease in maximal fat oxidation (32 ± 8 %) and a decreased maximal oxygen uptake (6 ± 2 %) were observed after regular excessive prolonged exercise (Authors unpublished findings). The blood glucose was unchanged, but the plasma FA concentration at rest was massively decreased by approx. 60 % after the 14 days. Muscle glycogen and triacylglycerol concentrations were unchanged after the 14 days. Muscle GLUT4 and HKII expression was increased after the 14 days and ATGL and LPL expression but not HSL expression, were also increased (Authors unpublished findings). Based on these data very prolonged exercise lead to a decreased maximal fat oxidation possibly due to a decreased exogenous fat availability. In a recent paper also based on the above described study, we demonstrated that energy expenditure during the 14 days were above 30 ± 2 MJ per day and although the body weight was unchanged there was a shift in body composition with a decreased body fat content (-2.2 +/- 0.7 kg and an increased lean mass (2.5 +/- 0.6 kg) (2). This implies that maximal fat oxidation after 14 days of prolonged exercise was decreased despite similar muscle glycogen and a sizeable energy deficit in these older men. Interestingly Slivka and colleagues observed a higher fat oxidation across a range of exercise intensities in 10 trained younger men after 21 days of very prolonged road cycling (4½ – 5 hours per day) (3). Clearly this difference may be explained by the differences in age, exercise load and the energy balance during the prolonged exercise. This talk will outline current knowledge on substrate utilization and muscle adaptation in ultra-endurance exercise and will with reference to the results of the study outlined above discuss possible mechanisms and future directions in this field.