Abstract: Signorile, JF, Lew, KM, Stoutenberg, M, Pluchino, A, Lewis, JE, and Gao, J. Range of motion and leg rotation affect electromyography activation levels of the superficial quadriceps muscles during leg extension. J Strength Cond Res 28(9): 2536–2545, 2014—Leg extension (LE) is commonly used to strengthen the quadriceps muscles during training and rehabilitation. This study examined the effects of limb position (POS) and range of motion (ROM) on quadriceps electromyography (EMG) during 8 repetitions (REP) of LE. Twenty-four participants performed 8 LE REP at their 8 repetition maximum with lower limbs medially rotated (TI), laterally rotated (TO), and neutral (NEU). Each REP EMG was averaged over the first, middle, and final 0.524 rad ROM. For vastus medialis oblique (VMO), a REP × ROM interaction was detected (p < 0.02). The middle 0.524 rad produced significantly higher EMG than the initial 0.524 rad for REP 6–8 and the final 0.524 rad produced higher EMG than the initial 0.524 rad for REP 1, 2, 3, 4, 6, and 8 (p ≤ 0.05). For rectus femoris (RF), EMG activity increased across REP with TO generating the greatest activity (p < 0.001). For vastus lateralis (VL), EMG increased across REP (p < 0.001) with NEU and TO EMG increasing linearly throughout ROM and TI activity greatest during the middle 0.524 rad. We conclude that to target the VMO, the optimal ROM is the final 1.047 rad regardless of POS, while maximum EMG for the RF is generated using TO regardless of ROM. In contrast, the VL is maximally activated using TI over the first 1.047 rad ROM or in NEU over the final 0.524 rad ROM.