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The high temperature mechanical property of separators is very important for safety of lithium-ion batteries. However, the mechanical integrity of polymeric separators in lithium-ion batteries at elevated temperatures is still not well characterized. In this paper, the temperature dependent micro-scale morphology change of PP (polypropylene)-PE (polyethylene)-PP sandwiched separators (Celgard 2325) was studied by in-situ high temperature surface imaging using an atomic force microscope (AFM) coupled with power spectral density (PSD) analysis and digital image correlation (DIC) technique. Both PSD and DIC analysis results show that the PP phase significantly closes its pores by means of dilation of the nanofibrils surrounding the pores in the transverse direction and shrinkage in the machine direction, when cycled at 90◦C, even below the separator’s shutdown temperature (∼120◦C) and its own melting temperature (165◦C). This is presumably due to surface melting effect in nanostructures and should be size dependent–the surface melting temperature may decrease with the diameter of nanofibrils. Therefore, some pore closing might happen even at operating temperatures, it will lead to capacity fade that is undesired for battery performance.