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Both stable isotope methodology and fluorescence microscopy were applied to define the use of intramuscular triglyceride (IMTG) stores as a substrate source during exercise on a whole‐body as well as on a fibre type‐specific intramyocellular level in trained male cyclists. Following an overnight fast, eight subjects were studied at rest, during 120 min of moderate intensity exercise (60 % maximal oxygen uptake capacity (V̇_O2,max)) and 120 min of post‐exercise recovery. Continuous infusions of [U‐¹³C]palmitate and [6,6‐²H₂]glucose were administered at rest and during subsequent exercise to quantify whole‐body plasma free fatty acid (FFA) and glucose oxidation rates and the contribution of other fat sources (sum of muscle‐ plus lipoprotein‐derived TG) and muscle glycogen to total energy expenditure. Fibre type‐specific intramyocellular lipid content was determined in muscle biopsy samples collected …