Pulsed Electric Field (PEF) pre-treatment, applied on fresh microalgae Auxenochlorella protothecoides, induces spontaneous release of a substantial water fraction and enables subsequent lipid extraction using ethanol-hexane blends. In this study, fresh microalgae suspensions were treated with PEF and incubated under inert conditions. Incubation promotes the release of ions and carbohydrates and increases the yields of subsequent lipid extraction thus enabling a considerable reduction of PEF-treatment energy. With a 20 h incubation period at 25 degrees C, almost total lipid extraction is achieved with a specific PEF-treatment energy of only 0.25 MJ/ kg(DW). Incubation on ice remains beneficial but less efficient than at 25 degrees C. Additionally, incubating microalgae cells in suspension at 100 g(DW)/L or in a dense paste, was almost equally efficient. Correlation between the different results suggests that spontaneous release of ions and carbohydrates facilitates more successful lipid extraction. A direct causality between the two phenomena remains to be demonstrated.