BACKGROUND Both training and normal body mass index are associated with high insulin sensitivity, but the mechanism may be different. OBJECTIVE The aim of the study was to examine whether lean trained humans may be protected from acute free fatty acid (FFA)-induced insulin resistance compared with lean sedentary humans. METHODS We conducted an interventional trial using either a 6-h lipid (20% Intralipid at 90 ml/h) or glycerol (2.25 g/100 ml at 90 ml/h) infusion along with a concurrent hyperinsulinemic-euglycemic clamp and serial muscle biopsies (0, 120, 360 min) at a clinical research unit at the University of Minnesota. METHODS The study included lean endurance-trained (n = 14) and sedentary (n = 14) individuals matched for age, gender, and body mass index. METHODS We measured the decline in glucose infusion rate (GIR) during the hyperinsulinemic-euglycemic clamp. RESULTS The trained group had higher baseline mitochondrial DNA copy number, mRNA of cytochrome C oxidase subunit 3, and insulin sensitivity (as measured by GIR) compared with the sedentary group. When FFA was acutely elevated to the upper physiological range (0.6-0.7 mEq/liter) by lipid infusion, the GIR in both activity groups declined similarly compared with their respective glycerol controls, although insulin signaling, as measured by Ser 473 pAKT/AKT, remained comparable. Specific to the trained group, the stimulatory effect of hyperinsulinemia on mitochondrial mRNA levels during the glycerol infusion was absent during the lipid infusion. CONCLUSIONS Elevated FFA had similar effects in reducing insulin sensitivity in trained and sedentary humans. In trained participants, this decline was associated with alterations in the skeletal muscle mitochondrial mRNA response to hyperinsulinemia.