The development of hemoglobin (Hb)-based oxygen carriers (HBOCs) has been hampered because of safety concerns in humans. Chemical and/or genetic modifications of the Hb introduce varied structural and conformational constraint on the molecule that resulted in proteins with diverse allosteric responses, nitrosative and oxidative side reactions. Here, we present for the first time a comprehensive biochemical and biophysical comparison of human, bovine, and genetically engineered HBOCs that have been tested in humans. We evaluate oxygen equilibrium and ligand binding kinetics under different experimental conditions as well as their autoxidation kinetics, redox reactions, and heme release. We determined the effects of HBOCs on cellular redox states and mitochondrial respiration. Taken together, these experiments provide a better understanding of the relationship between the structure-function and oxidative reactivity of these proteins. One can therefore select independently among these diverse properties to engineer a safe and effective HBOC with improved biochemical/biophysical characteristics.