Metabolic substrate utilisation of the human failing heart is an area of controversy. The purpose of this study is directly to quantify myocardial substrate utilisation in moderately severe heart failure, type 2 diabetes and healthy controls using simultaneous coronary sinus and arterial blood sampling. Patients with heart failure (n=9, mean NYHA 2.7±0.5), with type 2 diabetes (n=5) and with normal heart function (n=10) were studied after an overnight fast in connection with electrophysiological investigations/treatments. A systemic infusion of [2H2] palmitate allowed for the calculation of absolute palmitate extraction across the heart. Blood samples were analysed for non-esterified fatty acids, triacylglycerol, glycerol, glucose, pyruvate, lactate, 3-hydroxybutyrate and blood gases after simultaneous sampling of arterial and coronary sinus blood. Arteriocoronary sinus metabolite concentration differences and fractional extractions for all substrates were similar between the groups. The absolute non-esterified fatty acid uptakes assessed by [2H2]palmitate extraction were also similar between the groups. Using direct measurements of metabolic substrate uptake by arteriovenous difference technique, the compensated human failing heart does not appear to have reduced myocardial fatty acid uptake.