Trauma and sepsis are leading causes of death of intensive care patients. Injury triggers activation of innate immune system - e.g. peripheral blood mononuclear cells (PBMCs) or polymorphonuclear cells (PMNs) - possibly causing systemic inflammatory response syndrome (SIRS), leading to multiple organ dysfunction syndrome (MODS). Recent findings suggest that mitochondrial dysfunction is linked to the development of SIRS. The aim of the study was to develop a protocol to (i) isolate human white blood cells (WBCs) and (ii) measure mitochondrial respiration in WBCs. The application of the isolation protocol resulted in a yield of 38 % PBMCs and 44% PMNs. PMNs showed the lowest ROUTINE respiration and no oxygen consumption by ATP synthase. Reserve capacity was highest in PMNs followed by PBMCs. Compared to non-stimulated oxygen uptake, induction of inflammatory response by PMA resulted in a 7-fold increase in oxygen consumption accompanied by elevated reactive oxygen species generation. In summary, our results show that this protocol is appropriate to study mitochondrial function simultaneously with the measurement of respiratory burst. In future, we will apply this protocol to examine the impact of mitochondria targeted antioxidants on mitochondrial respiration of WBCs in sepsis in vivo models.