Aedes aegypti, the primary vector of various arthropod-borne viral (arboviral) diseases such as dengue and Zika, is a popular laboratory model in vector biology. However, its maintenance in laboratory conditions is difficult, mostly because the females require blood meals to complete oogenesis, which is often provided as sheep blood. The outermost layer of the mosquito cuticle is consists of lipids which protects against numerous entomopathogens, prevents desiccation and plays an essential role in signalling processes. The aim of this work was to determine how the replacement of human blood with sheep blood affects the cuticular and internal FFA profiles of mosquitoes reared in laboratory culture. The individual FFAs present in cuticular and internal extracts from mosquito were identified and quantified by GC-MS method. The normality of their distribution was checked using the Kolmogorov-Smirnov test and the Student’s t-test was used to compare them. GC-MS analysis revealed similar numbers of internal and cuticular FFAs in the female mosquitoes fed sheep blood by membrane (MFSB) and naturally fed human blood (NFHB), however MFSB group demonstrated 3.1 times greater FFA concentrations in the cuticular fraction and 1.4 times the internal fraction than the NFHB group. In the MFSB group, FFA concentration was 1.6 times higher in the cuticular than the internal fraction, while for NFHB, FFA concentration was 1.3 times lower in the cuticular than the internal fraction. The concentration of C18:3 acid was 223 times higher in the internal fraction than the cuticle in the MHSB group but was absent in the NFHB group. MFSB mosquito demonstrate different FFA profiles to wild mosquitoes, which might influence their fertility and the results of vital processes studied under laboratory conditions. The membrane method of feeding mosquitoes is popular, but our research indicates significant differences in the FFA profiles of MFSB and NFHB. Such changes in FFA profile might influence female fertility, as well as other vital processes studied in laboratory conditions, such as the response to pesticides. Our work indicates that sheep blood has potential shortcomings as a substitute feed for human blood, as its use in laboratory studies may yield different results to those demonstrated by free-living mosquitoes.