PURPOSE: To determine the physical characteristics that primarily influence tissue oxygenation assessments obtained by the Inspectra™ Tissue Spectrometer in the thigh and calf. METHODS: A total of 30 healthy men (age = 21.6 ± 1.6 yrs; BMI = 24.4 ± 3.5 kg/m2; mean ± SD) participated in this cross-sectional study. The Inspectra™ near-infrared spectrometer was used to assess tissue oxygenation in the thigh and calf after resting in a supine position for ten minutes. Subcutaneous adipose tissue thickness (SATT) was determined using ultrasound imaging (Fukuda Denshi 4500; Tokyo, Japan) with a 5 MHz transducer. Additionally, limb circumference was measured at the exact location of near-infrared spectroscopy assessments. The Johnson Space Center (NASA) Physical Activity Questionnaire (ranging from “0” to “7”) was used to score physical activity (PA) levels. RESULTS: A correlation matrix showed that tissue oxygenation values in the thigh were related (p < 0.05) to age (R = 0.390), BMI (R = −0.782), PA score (R = 0.428), thigh circumference (R = −0.673), and SATT (R = −0.909). The tissue oxygenation values in the calf were related (p < 0.05) to age (R = 0.406), PA score (R = 0.436), calf circumference (R = −0.341), and SATT (R = −0.497). Agraphical analysis ensured that no outliers were present. A multiple regression analysis was conducted for thigh (Adjusted r2 = 0.876) and the calf (Adjusted r2 = 0.379), where all significant correlates of tissue oxygenation were entered step-wise into the model. SATT was the primary predictor of tissue oxygenation values in the thigh (β = −0.611) and the calf (β = −0.440). PA score and BMI were also predictors in the thigh, while only PA score was an additional predictor in the calf. CONCLUSIONS: Our study shows that age, BMI, physical activity, limb circumference, and subcutaneous adiposity were all significantly related to lower limb tissue oxygenation. However, subcutaneous adiposity was the primary determinant of tissue oxygenation among these physical characteristics.