Background Recently, passive musculotendinous stiffness (MTS) has been assessed manually in the field; however, when conducting these types of assessments, the stretching velocity must be controlled to avoid eliciting the stretch reflex, which can be observed by increased electromyographic (EMG) amplitude of the stretched muscles and greater resistive torque (indicating the assessment is no longer passive). Objective To examine the effects of slow, medium, and fast stretching velocities during manually-applied passive straight-leg raise (SLR) assessments on hamstrings MTS and EMG amplitude characteristics. Study design Crossover study. Methods Twenty-three healthy, young adults underwent passive, manually-applied SLR assessments performed by the primary investigator at slow, medium, and fast stretching velocities. During each SLR, MTS and EMG amplitude were determined at 4 common joint angles (θ) separated by 5° during the final common 15° of range of motion for each participant. Results The average stretching velocities were 7, 11, and 18°·s−1 for the slow, medium, and fast SLRs. There were no velocity-related differences for MTS (P = 0.489) or EMG amplitude (P = 0.924). MTS increased (P < 0.001) with joint angle (θ1<θ2<θ3<θ4); however, EMG amplitude remained unchanged (P = 0.885) across the range of motion. Conclusions Although velocity discrepancies have been identified as a potential threat to the validity of passive MTS measurements obtained with manual SLR techniques, the present findings suggest that the SLR at any of the velocities tested in our study (7–18°·s−1) did not elicit a detectible stretch reflex, and thereby may be appropriate for examining MTS.