Intramyocellular lipid (IMCL) accumulation results in skeletal muscle insulin resistance (IR; 1). Intravenous lipid emulsion infusion in healthy humans during euglycaemic hyperinsulinaemia leads to impaired insulin stimulated whole body glucose uptake (2, 3), likely due to substrate competition (2) and/or accumulation of IMCL metabolites (1, 3). Skeletal muscle long-chain acylcarnitine (AC) accumulation from excess mitochondrial β-oxidation flux is associated with IR (4), but a direct link in vivo needs definitive evidence. Fish oil omega (n) 3 fatty acids can reduce lipid induced skeletal muscle IR in animal models (5). We hypothesised was that i.v. infusion of n3 lipid emulsion (Omegaven, Fresenius Kabi) in healthy humans will lessen the degree of IR normally observed with an n6 emulsion (Intralipid, Fresenius Kabi) due to reduced muscle accumulation of long-chain ACs. Six healthy men (age 26 ± 2 y, BMI 27 ± 2 kg/m2) participated in the present study, which was approved by the local ethics committee. On three randomised occasions at least one week apart subjects underwent a 6 h euglycaemic hyperinsulinaemic clamp (50 mU/m2/min) accompanied by infusion of saline (Con), 10% Intralipid (n6), or 10% Intralipid + 10% Omegaven (2:1; n3) at 100 ml/h. Arterialised-venous blood samples were taken hourly, and vastus lateralis muscle biopsy samples were taken before and after the clamp. Statistical analysis was performed using repeated-measures two-way ANOVA and data are expressed as means ± SEM. Steady state glucose disposal during the 6 h clamp was 28% lower in n6 compared to Con (57.3 ± 3.0 vs. 41.5 ± 2.6 nmol/kg/min; P<0.01). However, glucose disposal in n3 (51.4 ± 2.4 nmol/kg/min) was no different to Con, such that it was 24% greater than n6 (P<0.05). Skeletal muscle pyruvate dehydrogenase complex activation status (PDCa) increased during clamp in Con and n3 from 0.58 ± 0.08 to 1.15 ± 0.30 (P<0.01) and 0.73 ± 0.10 (P<0.05) mmol/kg/min wet mass, respectively, but remained unchanged in n6 (0.43 ± 0.09 mmol/kg/min wm). From a mean baseline content of 52.0 ± 0.1 μmol/kg dry mass, total AC (carbon lengths 3-20) content was supressed after the clamp in Con (21.3 ± 4.7 μmol/kg dm; P<0.001), but not in n6 or n3 (52.0 ± 14.3 and 49.6 ± 6.8 μmol/kg dm). However, whereas long-chain AC (12-20 carbons) was supressed by 54% (P<0.001), and there was a trend for muscle acetyl-CoA content to be 3-fold greater (P=0.09) in n6, they were unchanged in n3. In conclusion, i.v. n3 lipid infusion results in a reduced degree of lipid induced IR compared to equimolar n6 lipid infusion in healthy males. This effect was not due to a reduction in long-chain AC accumulation, but appears to be due to reduced shorter-chain mitochondrial β-oxidation flux resulting in a reduced degree of acetyl-CoA mediated inhibition of PDCa.