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SUMMARY: Background and aims: Preoperative fasting induces metabolic stress and leads to reduced postoperative insulin sensitivity, changes attenuated by preoperative carbohydrate loading. However, the mechanisms underlying these effects remain unknown. We investigated the dynamic changes in substrate metabolism and mononuclear cell mitochondrial function after fasting followed by refeeding with a drink [ONS (Fresenius Kabi, Germany)] designed to improve metabolic function preoperatively. Methods: Twelve healthy volunteers took part in this study. They were fed a standardized meal and studied 4h later (baseline ‘fed’ state), after 12 and 24h of fasting, and 2, 4 and 6h after ingestion of ONS (contained 100g carbohydrate, 30g glutamine, and antioxidants). Changes in liver and muscle glycogen and lipids were studied using 13C and 1H magnetic resonance spectroscopy. The activities of mitochondrial electron transport chain complexes I, II and IV in blood mononuclear cells were measured spectrophotometrically. Results: Compared to the baseline fed state, 12 and 24h fasts led to 29% and 57% decreases (P <0.001) in liver glycogen content, respectively. Fasting for 24h decreased mitochondrial membrane complexes I (−72%, P <0.05), II (−49%, P <0.01) and IV (−41%, P <0.05) activities compared to those following a 12h fast. A 23% increase (P <0.05) in calf intramyocellular lipid (IMCL) content occurred after a 24h fast. Liver glycogen reserves increased by 47% (P<0.05) by 2h following ingestion of ONS. Conclusions: Short-term fasting (up to 24h) affected mononuclear cell mitochondrial function adversely and increased IMCL content. Refeeding with ONS partially reversed the changes in liver glycogen. [ABSTRACT FROM AUTHOR]