Featured Application Importantly, measurements obtained through VOT-NIRS can vary significantly post-exercise, which should be considered by researchers before application.Abstract This study evaluated the influence of acute fatigue on heart rate variability (HRV) and muscle oxygen saturation (SmO2) at rest, as well as the reliability of SmO2 data measured using near-infrared spectroscopy (NIRS) during a vascular occlusion test (VOT). Twelve physically active subjects participated. Measurements included perceived muscle soreness using the visual analog scale (VAS pain), HRV parameters, variables of resting SmO2 (desaturation and resaturation), and reoxygenation kinetics (mean response time, MRT) through a VOT-NIRS located in the vastus lateralis (VL). Measurements were taken at three points: 24 h before, before exhaustive exercise, and 30 min after exhaustive exercise. The results indicated that acute fatigue increased resting muscle oxygen consumption in desaturation (+22 SmO2) and resaturation (+18 SmO2), improved MRT (-15 s), and elevated sympathetic nervous system (SNS) activity, as observed in the R-R interval (-262 ms) and SNS index (+0.5). HRV significantly influenced desaturation (r2 = 0.69), resaturation (r2 = 0.60), and MRT (r2 = 0.54). Reliability was established with an ICC of 0.49 and 0.63 for desaturation and resaturation, respectively. Real changes in desaturation and resaturation should be considered >= 7% SmO2 at rest and >= 11% SmO2 to avoid daily fatigue interference. In conclusion, acute fatigue increases resting SmO2 consumption and is associated with higher SNS activity and increased VAS pain.