Low-frequency fatigue was investigated in nine female and one male adult pigtail monkeys (Macaca nemestrina) with a mean weight of 5.3 kg (range 4.3 to 6.5 kg). After sedation and anesthesia, silver electrodes were inserted into the anterior and posterior parts of the right masseter muscle. The contralateral muscle was used as a control. The masseter muscles were stimulated for 3 minutes (4 Hz, 2 ms, 100 V). After a 5-minute rest period, the stimulation was repeated with the same duration and voltage but at a higher frequency of 8 Hz. Bite forces were measured, and muscle biopsies were obtained from the central part of the right masseter and immediately frozen in liquid nitrogen. After freeze-drying, a fluorometric analysis that used enzymatic methods for measuring levels of glycogen, glucose, lactate, pyruvate, creatine phosphate, nicotinamide-adenine dinucleotide (NAD), and reduced NAD (NADH) was performed. The bite force decreased by 12% after the initial 3 minutes of work. After the second contraction the bite force decreased to 56%. Prominent substrate depletion was observed. The precontraction levels of glycogen, glucose, and phosphocreatine were all reduced. The NADH and the NAD concentrations increased. An accumulation of metabolites was evident. The pyruvate increased by 32% and lactate levels increased by a factor of 3. The male measurements were comparable to the nine female measures for each assessment. The substantial substrate depletion in combination with a prominent accumulation of metabolites may contribute to the development of low-frequency fatigue.