In conclusion, we have synthesised an exo-methylenesubstituted pyrrolidinyl nitroxide iodoacetamide spin label with spirocyclohexyl substituents surrounding the paramagnetic centre. This label was successfully attached to a double mutant of calmodulin bound to the M13 peptide with high labelling efficiency. The protein-appended nitroxide showed high stability in the presence of excess ascorbate as a model system for a reducing environment. The lack of methyl group rotation resulted in long relaxation times up to 180 K, which enabled DEER measurements of interspin distance distributions even at elevated temperatures. These results provide a promising basis for the future application of spirocyclic pyrrolidinyl nitroxide spin labels in intracellular EPR measurements.