Abstract The thermal behaviour of phospholipid multilamellar vesicles (MLV) made of various molar percentages of DPPC and LPPC, containing also oxidized LPPC (LPPCox), was studied by use of EPR spectroscopy and n-DSPC spin label in order to determine variations in the membrane fluidity brought about by lipid oxidation. Experimental variables were temperature, ranging from 4 to 44 °C, and molar percentage composition of DPPC/LPPC/LPPCox ternary mixture. We found that the presence of LPPCox in a percentage higher than both normal phospholipids’ heavily hindered membrane formation, while lower percentage of the oxidized lipid with higher DPPC percentages yielded two-components EPR spectra, showing the presence of two different fluidity domains, indicative of membrane phase separation. When LPPC was the dominant lipid in the ternary mixture, simple EPR spectra were observed, indicating homogeneity of MLV membranes. Phase separation observed in the presence of LPPCox was better visible at lower temperature (12 °C or less), and almost disappeared with increasing temperature (36 °C or more). Furthermore, the correlation time of 16-DSPC in ternary mixture MLVs with higher LPPC percentage (homogeneous membranes) was not affected by the presence of LPPCox, while it normally increased upon DPPC percentage increase, as readily calculated from the EPR spectra featuring simple bands at 24 °C. It is concluded that oxidized lipid induces phase separation in more rigid DPPC-rich membranes, while leaving fluidity unaffected in more fluid LPPC-rich membranes, and at higher temperature.