Optical spectra were acquired from myoglobin and hemoglobin solutions and from the tibialis anterior muscle of Sprague–Dawley rats in the visible region (515 to 660 nm). Validation studies were performed on the <i>in vitro</i> spectra to demonstrate that partial least squares analysis of second-derivative spectra yields accurate measurements of myoglobin saturation in the presence of varying hemoglobin concentrations and saturations. When hemoglobin concentrations were varied between 0.25 and 4 times that of myoglobin, myoglobin saturations were measured with a root mean squared error (RMSE) of 4.9% (<i>n</i> = 56) over the full range from 0 to 1. Myoglobin saturations were also shown to be largely unaffected by hemoglobin saturation. RMSE values of only 1.7% (<i>n</i> = 77) were found when hemoglobin saturations were varied independently from myoglobin saturations. These <i>in vitro</i> validation studies represent the most complete and rigorous done to date using partial least squares analysis on myoglobin and hemoglobin spectra. Analysis of reflectance spectra from the rat hind limb yielded accurate measures of volume-averaged myoglobin fractional saturation in the presence of hemoglobin <i>in vivo</i>. Hemodilution showed that myoglobin fractional saturation measurements in the rat leg are not sensitive to changes in hematocrit, thereby confirming the results from solutions <i>in vitro</i>. Decreases in optical density of 11.3 ± 3.0% (<i>n</i> = 3) were achieved while myoglobin saturation decreased by only 3.1 ± 3.8%. Myoglobin saturation was significantly increased when the fraction of inspired O<sub>2</sub> was increased, showing that manipulations of myoglobin saturation are detectable and that myoglobin is not fully saturated in resting muscle. Together, these <i>in vitro</i> and <i>in vivo</i> studies show that cellular oxygenation derived from myoglobin fractional saturation can be measured accurately with little cross-talk from hemoglobin in the visible wavelength region, thereby extending optical spectroscopic studies of cellular and vascular oxygenation beyond the near-infrared regions previously studied.

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