Previous studies have used twitch electrical stimulation and accelerometer-based mechanomyography to evaluate muscle function in clinical populations. However, the reproducibility and validity of the methodology has not been defined. This studyevaluated the reproducibility and validity of twitch electrical stimulation and accelerometer-based mechanomyography (aMMG) as an assessment of muscle endurance. Participants were healthy males and females 21.8±1.9 years of age. Muscle twitch acceleration was measured using an accelerometer placed over the surface of the muscle. The relationship between acceleration and torque was measured during twitch stimulation of the vastus lateralis muscle. Muscle endurance of the forearm and gastrocnemius was measured during 9 minutes of twitch electrical stimulation, in three stages (3min/stage) of increasing frequency (2Hz, 4Hz, and 6Hz). An Endurance Index (EI) was calculated as the percent of acceleration at the end of each stimulation stage relative to the peak acceleration. Oxygen saturation was measured using near-infrared spectroscopy. Results showed that acceleration correlated with torque during twitch electrical stimulation of the vastus lateralis (mean R2= 0.96±0.04; p<0.05). Measures of forearm EI reproducibility were CV= 2.5-7.4%. EI was significantly higher (12.1%) in the gastrocnemius compared to the forearm (p<0.01). Muscle oxygen saturation was not reduced during stimulation of the forearm (72.6±9.8% at 2Hz, 73.2±11.6% at 4Hz, and 71.0±12.5% at 6Hz) compared to resting baseline (74.3±15.1%) (p>0.1). This study found that EI is a reproducible measure of muscle endurance that is not influenced by declines in oxygen saturation.