Context: 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. Design and Setting: 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 hyperinsulinemiceuglycemic clamp and serial muscle biopsies (0, 120, 360 min) at a clinical research unit at the University of Minnesota. Patients or Participants: The study included lean endurance-trained (n = 14) and sedentary (n = 14) individuals matched for age, gender, and body mass index. Main Outcome Measures: 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 FFAhadsimilar effects in reducing insulin sensitivity in trainedandsedentary humans. In trained participants, this decline was associated with alterations in the skeletal muscle mitochondrialmRNAresponse to hyperinsulinemia.