Muscle anabolic resistance to dietary protein is associated with obesity and insulin resistance. However, the contribution of excess consumption of fat to anabolic resistance is not well studied. The aim of these studies was to test the hypothesis that acute and short-term dietary fat overload will impair the skeletal muscle protein synthetic response to dietary protein ingestion. Eight overweight/obese males [46.4±1.4 years, BMI 32.3±5.4 kg/m2] participated in the acute feeding study, which consisted of 2 randomised crossover trials. On each occasion, subjects ingested an oral meal (with and without fat emulsion) 4h before the coingestion of milk protein, intrinsically labelled with [1-13C]phenylalanine, and dextrose. Nine overweight/obese males [44.0±1.7 years, BMI 30.1±1.1 kg/m2] participated in the chronic study, which consisted of a baseline 1-week isocaloric diet followed by a 2-week high fat diet (+25% energy excess). Acutely, incorporation of dietary amino acids into the skeletal muscle was 2-fold higher (P<0.05) in the lipid trial compared to control. There was no effect of prior lipid ingestion on indices of insulin sensitivity (muscle glucose uptake, PDC activity and Akt phosphorylation) in response to the protein/dextrose drink. Fat overfeeding had no effect on muscle protein synthesis or glucose disposal in response to whey protein ingestion, despite increased muscle DAG C16:0 (P=0.06) and ceramide C16:0 (P<0.01) levels. Neither acute nor short-term dietary fat overload has a detrimental effect on skeletal muscle protein synthetic response to dietary protein ingestion in overweight/obese men, suggesting dietary-induced accumulation of intramuscular lipids per se is not associated with anabolic resistance.