The concentration changes in oxygenated haemoglobin and deoxygenated haemoglobin in the brain cortex of guinea pigs associated with brain activation are measured from the multi-spectral images of the cortical tissue. The cortical tissue is observed through a thinned skull. The wavelength dependence of the optical path length is considered in the calculation of haemoglobin concentration. The results are compared with those obtained from the multi-spectral images of the exposed cortex to evaluate the influence of the thinned skull on the measurement of the concentration changes by multispectral imaging system. Although the skull thickness affected the sensitivity of the change in reflectance due to decrease in optical path in the cortical tissue, the influence of skull on the wavelength dependence of the optical path length can be ignored when the skull thickness is approximately less than 100 μm.