Near infrared spectroscopy (NIRS) noninvasively measures peripheral tissue oxygen saturation (StO2) and may be useful to detect early changes in StO2 in anaesthetized and critically ill horses. This study aimed to identify the muscle belly that provided the highest percentage of successful StO2 readings and the highest mean StO2 value. Fifty adult horses were enrolled in a prospective controlled study. StO2 was measured at six different muscles in each horse, for each intervention: hair overlying the muscle was clipped (post clipping: PC), clipped skin was cleaned with chlorhexidine (post-surgical prepping: PP) and medetomidine was administered intravenously (post medetomidine: PM). Mean StO2 values were calculated for each muscle, and a linear effects model was used to assess the effect of muscle group and intervention on StO2. The sartorius muscle gave the highest percentage of successful StO2 values (p < 0.001) and the highest mean (90% CI) StO2 values for the PC, PP and PM interventions. Surgical prepping of the skin increased the success for measurement of StO2 values. For all muscles, administration of medetomidine was associated with lower StO2 values (p < 0.001). In conclusion, of the muscles examined, the sartorius muscle may be the preferred muscle to measure StO2 in horses, and clipping and cleaning of the probe placement site is recommended.