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To evaluate in premature infants a new parenteral lipid emulsion based on olive and soybean oils (ratio 4:1), with less polyunsaturated fatty acids (PUFA) and more α-tocopherol than standard soybean oil emulsion.Premature infants (gestational age, 28–<37 weeks) were randomized to receive one of the two emulsions within the first 72 hours of life. The triglyceride dose was increased to 2 g/kg/day within 3 days. Plasma phospholipid fatty acids, α-tocopherol/lipid ratio, and urinary malondialdehyde (MDA) excretion were determined at baseline and after 7 days.Of 45 recruited infants, 33 completed the study per protocol (15 soybean oil, 18 olive oil emulsion). At study end, groups did not differ in plasma phospholipid arachidonic acid, total n-6 and n-3 metabolites, but the olive oil group showed higher values of the PUFA intermediates C18:3n-6 (0.19% ± 0.01% vs. 0.13% ± 0.02%, P < 0.05) and C20:3n-6 (2.92% ± 0.12% vs. 2.21% ± 0.17%, P = 0.005). The plasma α-tocopherol/total lipd ratio was higher in the olive oil group (2.45 ± 0.27 μmol/mmol vs. 1.90 ± 0.08 μmol/mmol, P = 0.001), whereas urinary MDA excretion did not differ.The lower PUFA supply with the olive/soybean oil emulsion appears to enhance linoleic acid conversion. The reduced PUFA content, combined with a higher antioxidant intake in the olive oil group, results in an improved vitamin E status. The olive oil-based emulsion is a valuable alternative for parenteral feeding of preterm infants who are often exposed to oxidative stress, while their antioxidative defense is weak.