Near-infrared spectrometry (NIRS) is a well-known method used to measure in vivo tissue oxygenation and hemodynamics. This method is used to derive relative measures of hemoglobin (Hb) + myoglobin (Mb) oxygenation and total Hb (tHb) accumulation from measurements of optical attenuation at discrete wavelengths. We present the design and validation of a new NIRS oxygenation analyzer for the measurement of muscle oxygenation kinetics. This design optimizes optical sensitivity and detector wavelength flexibility while minimizing component and construction costs. Using in vitro validations, we demonstrate 1) general optical linearity, 2) system stability, and 3) measurement accuracy for isolated Hb. Using in vivo validations, we demonstrate 1) expected oxygenation changes during ischemia and reactive hyperemia, 2) expected oxygenation changes during muscle exercise, 3) a close correlation between changes in oxyhemoglobin and oxymyoglobin and changes in deoxyhemoglobin and deoxymyoglobin and limb volume by venous occlusion plethysmography, and 4) a minimal contribution from movement artifact on the detected signals. We also demonstrate the ability of this system to detect abnormal patterns of tissue oxygenation in a well-characterized patient with a deficiency of skeletal muscle coenzyme Q₁₀. We conclude that this is a valid system design for the precise, accurate, and sensitive detection of changes in bulk skeletal muscle oxygenation, can be constructed economically, and can be used diagnostically in patients with disorders of skeletal muscle energy metabolism.