Understanding the thermal behavior of the solid-electrolyte interphase (SEI) is important for ensuring the overall safety and reliability of Li-ion batteries (LIBs). However, it is difficult to detect intrinsic exothermic heat flows from metastable SEI species during typical thermal characterization because of the high heat capacity of other cell components, such as active materials, binders, and current collectors, in samples. Here, we report a new approach to thermal and chemical characterization of the SEI on LIB anodes. By sampling the separator containing a top part of the SEI from cycled cells (graphite|LiCoO2), and conducting differential scanning calorimetry (DSC), X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy, we found that the SEI on the detached separator consisted mainly of metastable organic components, such as polycarbonates and Li alkyl carbonates, which were accumulated over repeated cycles and responsible for large amounts of exothermic signals at low temperatures, as shown in the DSC curves. These findings show that our sampling method enables a detailed thermal analysis on the metastable SEI alone, which can result in self-heating of cells under typical usage conditions.