For the working muscle there are a number of fuels available for oxidative metabolism, including glycogen, glucose and non-esterified fatty acids. Non-esterified fatty acids originate from lipolysis in white adipose tissue, from hydrolysis of VLDL-triglycerides or from hydrolysis of intramyocellular triglyceride stores. A key enzyme in the mobilization of fatty acids from intracellular lipid stores is hormone-sensitive lipase (HSL). The aim of the present study was to investigate the metabolic response of HSL-null mice challenged with exercise or fasting and to examine if other lipases are able to fully compensate for the lack of HSL. The results showed that HSL-null mice have reduced capacity to perform aerobic exercise. The liver glycogen stores were more rapidly depleted in HSL-null mice during treadmill exercise and HSL-null mice had reduced plasma concentrations of both glycerol and non-esterified fatty acids after exercise and fasting, respectively. The data support the hypothesis that in the absence of HSL mice are not able to respond to an exercise challenge with increased mobilization of the lipid stores. Consequently, the impact of the lipid sparing effect on liver glycogen will be reduced in the HSL-null mice, resulting in faster depletion of this energy source, contributing to the decreased endurance during sub-maximal exercise.