Our study investigates short- and long-term effects of infusion of non-esterified fatty acids (NEFA) on insulin secretion in healthy subjects. Twelve healthy individuals underwent a 24-h Intralipid (10% triglyceride emulsion) infusion at a rate of 0.4 ml/min with a simultaneous infusion of heparin (a bolus of 200 U followed by 0.2 U/min per kg body weight). After an overnight fast (baseline), at 6 and at 24 h of Intralipid infusion and 24 h after Intralipid discontinuation (recovery test), all subjects underwent an intravenous glucose tolerance test (iv-GTT) (25 g of glucose/min). Intralipid infusion caused a threefold rise in plasma NEFA concentrations with no difference between the 6- and the 24-h concentrations. Compared to baseline acute insulin response (AIR) (AIR=63±8 mU/l), short-term (6-h) Intralipid infusion was associated with a significant increase in AIR (86±12 mU/l p<0.01); in contrast, long-term (24-h) Intralipid delivery was associated with inhibition of AIR (31±5 mU/l) compared to baseline (p<0.001) and to the 6-h (p<0.03) triglyceride emulsion infusion. Intralipid infusion was associated with a progressive and significant decline in respiratory quotient (RQ). A positive correlation between changes in fasting plasma NEFA concentrations and AIR at the 6-h infusion (r=0.89 p<0.001) was found. In contrast, at the end of the Intralipid infusion period, changes in plasma NEFA concentrations and AIR were negatively correlated (r=−0.87 p<0.001). The recovery test showed that fasting plasma NEFA concentrations, RQ and AIR had returned to baseline values. In the control study (n=8) 0.9% NaCl infusion did not mimick the effect of Intralipid. In conclusion, our study demonstrates that short- and long-term exposures of beta cells to high plasma NEFA concentrations have opposite effects on glucose-induced insulin secretion.