Background: Mixed lipid emulsions (LE) containing fish oil present several advantages compared to the sole soybean oil LE, but little is known about the safety of essential fatty acids (EFA) profile in paediatric patients on long-term Parenteral Nutrition (PN). Aim of the study: to assess glycerophosfolipid polyunsaturated fatty acids (PUFA) levels on plasma and red blood cell (RBC) membrane of children on long term PN with composite LE containing fish oil (SMOF), and to compare it with a group receiving olive oil LE (Clinoleic®) and to the reference range for age, previously determined on a group of healthy children. Results: A total of 38 patients were enrolled, median age 5.56 (0.9-21.86) years, 15 receiving Clinoleic®, 23 receiving SMOF. Patients on SMOF showed significantly higher levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), lower levels of arachidonic acid (ARA) and Mead acid (MEAD)/ARA ratio in plasma and RBC compared with patients on Clinoleic® and with healthy children. Triene:tetraene (T:T) ratio of both groups of patients did not differ from that of healthy children-median plasma (MEAD/ARA: 0.01, interquartile rage (IQR) 0.01, p = 0.61 and 0.02, IQR 0.02, p = 0.6 in SMOF and Clinoleic® patients, respectively), and was considerably lower than Holman index (>0.21). SMOF patients showed no statistically significant differences in growth parameters compared with Clinoleic® patients. Patients of both groups showed stiffness class F0-F1 of liver stiffness measure (LSM) 5.6 (IQR 0.85) in SMOF patients and 5.3 (IQR 0.90) in Clinoleic® patients, p = 0.58), indicating absence of liver fibrosis. Conclusions: Fatty acids, measured as concentrations (mg/L), revealed specific PUFA profile of PN patients and could be an accurate method to evaluate nutritional status and eventually to detect essential fatty acid deficiency (EFAD). SMOF patients showed significantly higher EPA, DHA and lower ARA concentrations compared to Clinoleic® patients. Both LEs showed similar hepatic evolution and growth.