Introduction: The tissue hemoglobin index (THI) is a hemoglobin signal strength metric provided on the InSpectra™ StO 2 Tissue Oxygenation Monitor, Model 650. There is growing interest regarding the physiologic meaning of THI and whether a clinically useful correlation between THI and blood hemoglobin concentration exists. A series of in vitro and in vivo experiments was performed to evaluate whether THI has potential utility beyond its primary purpose of helping InSpectra™ device users optimally position a StO 2 sensor over muscle tissue.Methods: The THI and tissue hemoglobin oxygen saturation (StO 2) were measured using the InSpectra™ StO 2 Tissue Oxygenation Monitor, Model 650, with a 15 mm optical sensor. A THI normal reference range was established in the thenar eminence (hand) for 434 nonhospitalized human volunteers. In 30 subjects, the thenar THI was also evaluated during 5-minute arterial and venous blood flow occlusions, and with blood volume exsanguination in the hand induced with an Esmarch bandage. In addition, correlation of the THI to blood total hemoglobin concentration (Hbt) was studied in five pigs whose Hbt was isovolumetrically diluted from 13 to 4 g/dl systemically and 0.5 g/dl locally in the hind limb. The sensitivity and specificity of the THI to measure tissue hemoglobin concentration (THC) were characterized in vitro using isolated blood tissue phantoms.Results: In human thenar tissue, the average THI was 14.1 ± 1.6 (mean ± standard deviation). The THI extrapolated to 100% blood volume exsanguination was 3.7 ± 2.0 units presumably from myoglobin. On average, the THI increased 1.5 ± 1.0 units with venous occlusion and decreased 4.0 ± 2.0 units with arterial occlusion. In porcine hind limbs, the THI weakly correlated with Hbt (r 2 = 0.26) while ΔTHI during venous occlusion had a stronger correlation (r 2 = 0.62). In vitro tests indicated that THI strongly correlated (r 2 > 0.99) to phantom THC and was insensitive to StO 2 changes.Conclusions: Steady-state THI values do not reliably indicate Hbt. The THI is a reproducible quantitative index for THC, and THI trends can discriminate between arterial or venous blood flow occlusions. The THI magnitude permits the estimation of myoglobin's contribution to StO 2.