This study reports a novel protocol to increase plasma monounsaturated, polyunsaturated and saturated non-esterified fatty acids (NEFA) in eight healthy volunteers (age 29-54 years and BMI 23-26 kg.m^-2). This was achieved by feeding small boluses of fat at different time points (35 g at 0 min and 8 g at 30, 60, 90, 120, 150, 180, 210 min) in combination with a continuous low-dose heparin infusion. Olive oil, safflower oil or palm stearin were used to increase monounsaturated, polyunsaturated or saturated NEFAs, respectively. Plasma NEFA concentrations were increased for 2 h, when fat and heparin were given (olive oil: 745 ± 35 µmol.l^-1; safflower oil: 609 ± 37 µmol.l^-1 and palm stearin: 773 ± 38 µmol.l^-1) compared to the control test (no fat and no heparin: 445 ± 41 µmol.l^-1). During the heparin infusion, 18:1n-9 was the most abundant fatty acid for the olive oil test, compared to 18:2n-6 for the safflower oil test and 16:0 for the palm stearin test (P<0.01). The method described here, successfully increases several types of plasma NEFA concentrations and could be used to investigate differential effects of elevated individual NEFAs on metabolic processes.