Mitochondrial bioenergetics of PBMCs have been linked with several factors that contribute to a better understanding of several human diseases. Due to the complex logistics of clinical studies, samples are often cryopreserved for later analysis. Current data on whether cryopreservation negatively affects the mitochondrial function of PBMCs is discrepant. We isolated and cryopreserved peripheral blood mononuclear cells (PBMCs) from human whole blood and tested mitochondrial function using a substrate-uncoupler-inhibitor-titration protocol on the Oroboros instrument. After three months of storage in a cryopreservation medium (at -80 °C), several aspects of mitochondrial bioenergetics were measured. We demonstrate that cryopreservation did not adversely affect mitochondrial parameters (routine, leak, complex-I linked OXPHOS, cytochrome-c response, ETS capacity, the contributions of the N and S-pathways to ETS, ROX, complex-IV activity and mitochondrial coupling). Therefore, after three months of cryopreservation at -80 °C, human PBMC-mitochondria were fully coupled and functional. Therefore, clinical studies may cryopreserve PBMCs for later mitochondrial analyses.