Key points Although lipid droplets in skeletal muscle are an important energy source during endurance exercise, our understanding of lipid metabolism in this context remains incomplete. Using transmission electron microscopy, two distinct subcellular pools of lipid droplets can be observed in skeletal muscle – one beneath the sarcolemma and the other between myofibrils. At rest, well‐trained leg muscles of cross‐country skiers contain 4‐ to 6‐fold more lipid droplets than equally well‐trained arm muscles, with a 3‐fold higher content in type 1 than in type 2 fibres. During exhaustive exercise, lipid droplets between the myofibrils but not those beneath the sarcolemma are utilised by both type 1 and 2 fibres. These findings provide insight into compartmentalisation of lipid metabolism within skeletal muscle fibres. Abstract Although the intramyocellular lipid pool is an important energy store during prolonged exercise, our knowledge concerning its metabolism is still incomplete. Here, quantitative electron microscopy was used to examine subcellular distribution of lipid droplets in type 1 and 2 fibres of the arm and leg muscles before and after 1 h of exhaustive exercise. Intermyofibrillar lipid droplets accounted for 85–97% of the total volume fraction, while the subsarcolemmal pool made up 3–15%. Before exercise, the volume fractions of intermyofibrillar and subsarcolemmal lipid droplets were 4‐ to 6‐fold higher in leg than in arm muscles (P < 0.001). Furthermore, the volume fraction of intermyofibrillar lipid droplets was 3‐fold higher in type 1 than in type 2 fibres (P < 0.001), with no fibre type difference in the subsarcolemmal pool. Following exercise, intermyofibrillar lipid droplet volume fraction was 53% lower (P = 0.0082) in both fibre types in arm, but not leg muscles. This reduction was positively associated with the corresponding volume fraction prior to exercise (R2 = 0.84, P < 0.0001). No exercise‐induced change in the subsarcolemmal pool could be detected. These findings indicate clear differences in the subcellular distribution of lipid droplets in the type 1 and 2 fibres of well‐trained arm and leg muscles, as well as preferential utilisation of the intermyofibrillar pool during prolonged exhaustive exercise. Apparently, the metabolism of lipid droplets within a muscle fibre is compartmentalised.