Although elevated free fatty acid (FFA) levels in obesity have been considered to be of importance for insulin resistance, a recent meta-analysis suggested normal FFA levels in obese subjects. We investigated fasting circulating FFA and glycerol levels in a large cohort of non-obese and obese subjects. Subjects recruited for a study on obesity genetics were investigated in the morning after an overnight fast (n= 3,888). Serum FFA (n= 3,306), plasma glycerol (n= 3,776), and insulin sensitivity index (HOMA-IR, n= 3,469) were determined. Obesity was defined as BMI≥ 30 kg/m 2 and insulin resistance as HOMA-IR≥ 2.21. In obese subjects, circulating FFA and glycerol levels were higher than in non-obese individuals (by 26% and 47%, respectively; both p< 0.0001). Similar results were obtained if only men, women or medication-free subjects were investigated. Insulin resistance and type 2 diabetes were associated with a further minor increase in FFA/glycerol among obese subjects. When comparing insulin-sensitive non-obese with insulin-sensitive or-resistant obese individuals, FFA and glycerol were 21-29% and 43-49% higher in obese individuals, respectively. Circulating FFA and glycerol levels are markedly elevated in obesity but only marginally influenced by insulin resistance and type 2 diabetes. Whether these differences persist during diurnal variations in circulating FFA/glycerol, remains to be established.