Diffuse reflectance spectroscopy using fiber optic probe is one of most promising technique for evaluating optical properties of biological tissue. We present a method determining the reduced scattering coefficients μs’, the absorption coefficients μ a, and tissue oxygen saturation StO2 of in vivo brain tissue using single reflectance fiber probe with two source-collector geometries. In the present study, we performed in vivo recordings of diffuse reflectance spectra and the electrophysiological signals for exposed brain of rats during normoxia, hyperoxia, hypoxia, and anoxia. The time courses of μa in the range from 500 to 584 nm and StO2 indicated the hemodynamic change in cerebral cortex. Time courses of μs’ are well correlated with those of μa in the range from 530 to 570 nm, which also reflect the scattering by red blood cells. On the other hand, a fast decrease in μs’ at 800 nm were observed after the respiratory arrest and it synchronized with the negative deflection of the extracellular DC potential. It is said that the DC shift coincident with a rise in extracellular potassium and can evoke cell deformation generated by water movement between intracellular and extracellular compartments, and hence the light scattering by tissue. Therefore, the decrease in μs’ at 800 after the respiratory arrest is indicative of changes in light scattering by tissue. The results in this study indicate potential of the method to evaluate the pathophysiological and loss of tissue viability in brain tissue.