Near infrared spectroscopy (NIRS) can monitor both the redox status of Cytochrome c oxidase located in the mitochondria within the cell and the oxygenation of the blood in the tissue being monitored. Since the enzyme catalyzes more than 90% of oxygen utilization, it is the sink for the oxygen while the hemoglobin in the capillaries is the oxygen source. In order to evaluate the oxidative metabolic status of a tissue the optical data obtained from both molecules are commonly interpreted on the basis of test tube experiments with purified preparations. We are concerned that the validity of this practice may not have been tested sufficiently and raise four basic questions that have not yet been answered. Citing some examples of in vitro versus in vivo differences we conclude that more effort should be expended on the in vivo testing of the range of the signals, their natural variability, and the physiological and pathological meaning of their deviations from norm.