We analyze the interface trap states generated by the self-heating effect in flexible single-crystalline Si nanomembrane (sc-Si NM) transistors. Despite the excellent device performance (Subthreshold swing: ~61 mV/dec, Ion/off: ~109, Nit: ~5 × 1010 cm-2, µeff: ~250 cm²/V·s) and mechanical flexibility (RB,min ═ 1 mm) of sc-Si NM transistors on a polymer substrate, they are vulnerable to thermal reliability issues due to the poor thermal conductivity (κ < 1 W/m·K) of the polymer substrate. Understanding the detailed mechanism driving heat-related device degradation is key to improving device reliability, life expectancy, and overall device performance. Thus, a charge pumping method was employed to systematically analyze the device degradation caused by the self-heating effect. This enabled the interface trap density to be investigated for the flexible sc-Si NM transistors on a polymer substrate after a bias stress. For comparison, a heat spreading layer (HSL) made using a 1-µm thick Ag film (κ~400 W/m·K) was integrated into the sc-Si NM device to mitigate the self-heating effect. The results showed that the interface trap density was proportional to the self-heating effect. This facilitated the fundamental understanding of the self-heating effect of flexible sc-Si NM transistors, opening a robust route to realizing high performance flexible devices using sc-Si NM.