Introduction: Anatomical properties between proximal and other regions within the human rectus femoris (RF) muscle are nonuniform. We aimed to clarify the possible region-specific myoelectric manifestations of fatigue within the RF muscle by using an advanced surface electromyography (SEMG) technique. Methods: Nine healthy men performed sustained contractions at 50% of maximal voluntary contraction until exhaustion during isometric knee extension and hip flexion. During these contractions, multi-channel SEMG was recorded from the RF by using 46 electrode pairs which cover most of the superficial area of the muscle. Results: Fatigue-induced SEMG, i.e., an increase in root mean square and a decrease in median frequency, was not uniform within the muscle during both tasks and was greater in proximal regions. Conclusions: Our findings suggest that myoelectric manifestations of fatigue within the human RF muscle are localized, and proximal regions are more fatigable than other regions within this muscle.