Skeletal muscle insulin resistance associated with ageing may result from age-related intramyocellular lipid (IMCL) accumulation. Whether IMCL derived fatty acid oxidation is impaired during exercise in older individuals is unclear. Moreover, there are disctinct pools of IMCL droplets localised to intermyofibrillar (IMF) and subsarcolemmal (SSL) regions of the myofibre, and little is known about the relative contribution of these pools to lipid use during exercise. To answer these questions, 14 healthy older (69 ± 1 yr, BMI 26.5 ± 0.8 kg/m2) and 8 younger (22 ± 1 yr, BMI 24.0 ± 1.1 kg/m2) men performed 1 hr of cycling at 50% maximal oxygen consumption and the contribution of plasma fatty acids and intramyocellular lipid (intermyofibrillar and subsarcolemmal) to total fat oxidation was determined using a combination of indirect calorimetry, intravenous [U-13C]palmitate infusion, and electron microscopy of muscle biopsies. The study was approved by the University of Nottingham Medical School Ethics Committee. Two-way ANOVA was performed to detect within and between groups differences, with statistical significance declared at P<0.05 (all values represent mean ± standard error). There was no difference in the relative contribution of fat to total energy expenditure during exercise when comparing older and younger volunteers (41.2 ± 2.7 vs. 43.3 ± 6.1%, respectively), but the relative contribution of IMCL to total fat oxidation was 40% less in older subjects (42.0 ± 6.0 vs. 69.9 ± 3.5%, respectively; P<0.01). This difference was not attributable to reduced utilisation of IMF lipid droplets as the decrease in the % of IMF fibre area occupied by droplets after exercise was similar in between younger (0.65 ± 0.11 to 0.40 ± 0.09%; P<0.05) and old er(0.72 ± 0.09 to 0.57 ± 0.12%; P=0.07) subjects, and was predominantly due to a reduction in droplet number (22.2 ± 3.0 to 15.4 ± 2.7 and 23.3 ± 2.6 to 17.4 ± 2.9 droplets/mm2 fibre for younger and older subjects, respectively; P<0.05). Indeed, average droplet size was unchanged by exercise in both age groups. Conversely, in the SSL region average droplet size (0.32 ± 0.04 to 0.40 ± 0.05 µm2; P<0.05) increased in older, but not younger, subjects after exercise, such that the relative SSL fibre area occupied by droplets was ~3-fold greater in older compared to younger subjects (3.55 ± 0.66 vs. 1.26 ± 0.27%; P<0.05). Furthermore, the difference between rates of plasma fatty acid disappearance and oxidation was ~2 fold greater in older vs. younger subjects (16.2 ± 2.2 vs. 9.6 ± 1.7 µmol/kg lean mass/min, respectively; P<0.05). In conclusion, IMCL utilisation during exercise is blunted by age, which is localised to the SSL region and attributable to an imbalance between plasma fatty acid delivery and oxidation. SSL IMCL accumulation may explain age associated insulin resistance, particularly as this is the site of insulin action and glucose uptake.