Highlights •TIRF microscopy enabled the tracking of the plasma membrane lipid oxidation.•Oxidation was measured in PEF-treated cells placed on conductive ITO coverslips.•µs- but not nsPEFs caused oxidation at permeabilizing E-fields in Tyrode’s solution.•Decreasing medium conductivity enhanced permeabilization but suppressed oxidation.•Such low levels of oxidation could not account for persistent permeabilization.