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INTRODUCTION The purpose of this study was to examine the acute effect of different rest intervals between multiple sets of the 45° angled leg press exercise (LP45) on surface electromyographic (SEMG) spectral and amplitude indices of fatigue. METHODS Fifteen recreationally trained females performed three protocols in a randomized crossover design; each consisting of four sets of 10 repetitions with 1 (P1), 3 (P3), or 5 (P5) minute rest intervals between sets. Each set was performed with 70% of the LP45 ten-repetition maximum load. The SEMG data for biceps femoris (BF), vastus lateralis (VL), vastus medialis (VM), and rectus femoris (RF) muscles was then evaluated. RESULTS The SEMG amplitude change in the time coefficient (CRMS) and spectral fatigue index (Cf5) indicated higher levels of fatigue for all muscles evaluated during the P3 protocol versus the P1 and P5 protocols (p ≤ 0.05), respectively. The RF and VL muscles showed greater fatigue levels by the second and third sets; whereas, greater fatigue was shown in the VM and BF muscles by the fourth set (p ≤ 0.05). CONCLUSIONS A three-minute rest interval between sets might represent a neuromuscular window between a fatigue stated and fully recovered state in the context of neural activation. Moreover, a three minute rest interval between sets might allow for consistent recruitment of high threshold motor units over multiple sets, and thus promote a more effective stimulus for strength gains.