This letter presents an experiment to explore the nonisothermal effects on temperature and emissivity separation (TES). The innovation of this experiment lies in its design, which highlights the contrast between isothermal and nonisothermal conditions in emissivity measurements. We artificially created a sharply contrasting nonisothermal soil surface using liquid nitrogen cooling and solar heating. The iterative spectrally smooth TES (ISSTES) algorithm was used to process the experimental data. The analyzed results of the experimental data show that the nonisothermal conditions have a significant effect on TES. The bias of retrieved emissivity increases with the component temperature difference as well as with wavelength. The bias around the split window band can reach up to 1% when the difference of the component temperature is 40K. Considering that 1% error in emissivity can cause approximately 1K error of retrieved land surface temperature (LST), the nonisothermal effects on emissivity cannot be ignored. We hope that this experiment will arouse attention of the nonisothermal effects on TES and call for more efforts to be devoted to this issue in the future.