Obesity and insulin resistance are associated with an impaired sensitivity to anabolic stimuli such as dietary protein (anabolic resistance). Omega-3 polyunsaturated fatty acids (n-3 PUFA) may be protective against the deleterious effects of saturated fatty acids (SFA) on insulin resistance. However, the contribution of excess fat consumption to anabolic and insulin resistance and the interaction between SFA and n-3 PUFA is not well studied. The primary aim of this study was to investigate the effects of an oral fat pre-load, with or without the partial substitution of SFA with fish oil (FO)-derived n-3 PUFA, on indices of insulin and anabolic sensitivity in response to subsequent dietary protein and carbohydrate (dextrose) co-ingestion. Eight middle-aged males with overweight or obesity (52.8±2.0yr, BMI 31.8±1.4kg·m-2) ingested either an SFA, or isoenergetic SFA and FO emulsion (FO), or water/control (Con), 4h prior to a bolus of milk protein and dextrose. Lipid ingestion (in particular FO) impaired the early postprandial uptake of branched chain amino acids (BCAA) into the skeletal muscle in response to protein and dextrose, and attenuated the peak glycaemic response, but was not accompanied by differences in whole body (Matsuda Index: Con: 4.66±0.89, SFA: 5.10±0.94 and FO: 4.07±0.59) or peripheral (forearm glucose netAUC: Con: 521.7±101.7; SFA: 470.2±125.5 and FO: 495.3±101.6μmol·min-1·100g lean mass·min [t=240-420min]) insulin sensitivity between visits. Postprandial whole body fat oxidation was affected by visit (P=0.024) with elevated rates in SFA and FO, relative to Con (1.85±0.55; 2.19±0.21 and 0.65±0.35kJ·h-1·kg-1 lean body mass, respectively), however muscle uptake of free fatty acids (FFA) was unaffected. Oral lipid preloads, consisting of SFA and FO, impair the early postprandial BCAA uptake into skeletal muscle, which occurs independent of changes in insulin sensitivity. ClinicalTrials.gov Identifier NCT03146286.