This paper examined the role of metabolites in causing muscle fatigue. Previous studies have shown that Pi [Formula: see text] and H+ may be important factors in causing fatigue. A key question is the potential interaction between metabolic end-products and calcium related excitation-contraction coupling fatigue (ECC). An in vivo rat muscle model was used to measure tension development and metabolic end-products in response to electrical stimulation. Two stimulation protocols were used, high intensity stimulation followed by a medium intensity stimulation (High Group), and low intensity stimulation followed by a medium intensity stimulation (Low Group). Metabolic fatigue was based on concentrations of [Formula: see text] measured with phosphorus magnetic resonance spectroscopy. ECC fatigue was measured as the fatigue in excess of metabolic fatigue, and as the relative decline of force at low compared to high stimulation frequencies. During the initial stimulation period, the High Group had greater metabolic fatigue (p < 0.001) and greater ECC fatigue (p = 0.007). During the second stimulation period and recovery, the High Group had no difference in metabolic fatigue (p = 0.07) and greater ECC fatigue (p = 0.015). These results present a method for determining the relative amounts of metabolic and ECC fatigue, and suggest that metabolites can increase the amount of ECC fatigue. Key words: fatigue, skeletal muscle, excitation contraction coupling