We have further developed and refined our EPR technique for in vivo temperature measurements. The response of the nitroxide stable free radicals has been substantially enhanced by encapsulating them in a medium of a fatty acid mixture inside a protein microsphere. The mixture undergoes a phase transition in the temperature range required by the application. The phase change dramatically alters the shape of the EPR spectrum, providing a highly temperature sensitive signal. Using the nitroxide dissolved in a cholesterol and a long-chain fatty acid ester, we developed a mixture which provides a peak-height ratio change from 3.32 to 2.11, with a standard deviation of 0.04, for a temperature change typical in biological and medical applications, from 37 to 47 °C. This translates to an average temperature resolution of 0.2 °C. The average diameter of the nitroxide mixture filled microspheres is ≈2 mm. Therefore, they are compatible with in vivo studies where the microspheres are injected into the microvasculature having a minimum vessel diameter of the order of 10 mm. We are currently examining the simulation of the EPR spectra to improve temperature sensitivity as well as to develop tomographic techniques. This temperature measuring method has various, potential clinical applications, especially in monitoring and optimizing the treatment of cancer with hyperthermia.