Spatially resolved spectroscopy (SRS) allows the estimation of absolute tissue oxygen saturation, the ratio of oxygenated to total hemoglobin concentration, which may facilitate the comparison of results among patients. Eighty-two premature infants were included over two years. The cerebral tissue oxygenation index (c-TOI) was measured using NIRO 300 (Hamamatsu Photonics KK). c-TOI was measured at several positions in each infant. c-TOI varied over time, increasing in the first third and decreasing in the last third of the study period . Two probes were used in the study, and a highly significant difference was found between these . The mean difference was 8.5% (95%CI 5.4 to 11.6%). After correction for this difference, there was no variation over time. A conclusive explanation for the bias could not be identified. Since the study groups were well distributed, the bias had no influence on the results of our clinical study. We investigated an unexpected but highly significant probe-dependent bias in c-TOI with no conclusive explanation. Hence, comparisons of absolute TOI between groups of patients and among studies should be regarded with caution. A better strategy to detect potential instrumental problems will be useful in preventing biased c-TOI from occurring.