The reliability of MyotonPRO that can monitor the mechanical properties of tissues is still unclear. This study aimed to analyze the within-day inter-operator and between-day intra-operator reliability of MyotonPRO for assessing tone and stiffness of quadriceps femoris and patellar tendon at different knee angles. The tone and stiffness of healthy participants (15 males and 15 females, aged 24.7±1.6 years) in the supine and resting position were measured using the MyotonPRO device. The measurements were quantified at 0, 30, 60, and 90 of knee flexion. The intraclass correlation coefficient (ICC), standard error of measurement (SEM), and minimal detectable change (MDC) were calculated and a Bland–Altman analysis was conducted to estimate reliability. The results indicated excellent inter-operator reliability (ICC > 0.78) and good to excellent intra-operator reliability (ICC > 0.41). The inter-operator SEM measurements ranged between 0.1–0.9 Hz and 3.8–37.9 N/m, and intra-operator SEM ranged between 0.5–1.3 Hz and 7.9–52.0 N/m. The inter-operator MDC ranged between 0.3–2.5 Hz and 10.5–105.1 N/m, and intra-operator SEM ranged between 1.1–3.3 Hz and 21.9–144.1 N/m. The agreement of inter-operator was better than that of intra-operator. The study concluded that MyotonPRO is a reliable device to detect the tone and stiffness of quadriceps femoris and patellar tendon. © 2019 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.