To develop and evaluate a two-dimensional (2D) fast spin echo (FSE) pulse sequence for enhancing temporal resolution and reducing tissue heating for in vivo proton electron double resonance imaging (PEDRI) of mice. A four-compartment phantom containing 2 mM TEMPONE was imaged at 20.1 mT using 2D FSE-PEDRI and regular gradient echo (GRE)-PEDRI pulse sequences. Control mice were infused with TEMPONE over ~1 min followed by time-course imaging using the 2D FSE-PEDRI sequence at intervals of 10-30 s between image acquisitions. The average signal intensity from the time-course images was analyzed using a first-order kinetics model. Phantom experiments demonstrated that EPR power deposition can be greatly reduced using the FSE-PEDRI pulse sequence compared with the conventional gradient echo pulse sequence. High temporal resolution was achieved at ~4 s per image acquisition using the FSE-PEDRI sequence with a good image SNR in the range of 233-266 in the phantom study. The TEMPONE half-life measured in vivo was ~72 s. Thus, the FSE-PEDRI pulse sequence enables fast in vivo functional imaging of free radical probes in small animals greatly reducing EPR irradiation time with decreased power deposition and provides increased temporal resolution.