BACKGROUND The aim of this study was to assess the passive stiffness of the medial and lateral gastrocnemius (MG and LG), Achilles tendon (AT), and plantar fascia (PF) at different ankle and knee positions. MATERIAL AND METHODS Stiffness was assessed using a portable hand-held device (MyotonPRO). In 30 healthy participants (15 males, 15 females) with the knee fully extended or flexed 90°, stiffness of the MG, LG, AT, and PF was measured at 50° plantar flexion, 0° (neutral position), and 25° dorsiflexion (not for AT) of the ankle joint by passive joint rotation. RESULTS With the knee fully extended, passive dorsiflexion caused significant increase in muscle stiffness (P<0.001), whereas AT and PF stiffness increased with passive ankle dorsiflexion regardless of knee position (P<0.001). Increased stiffness was observed in MG compared to LG (P<0.001) and at the 3-cm site of AT compared to the 6-cm site (P<0.05). Stiffness was greater in LG compared to MG at -50° plantar flexion (P<0.001) and was greater in MG compared to LG at 25° dorsiflexion (P<0.05). Stiffness of AT increased in a distal-to-proximal pattern: 0 cm >3 cm >6 cm (P<0.001). CONCLUSIONS Stiffness assessed by use of the MyotonPRO was similar assessments using other techniques, suggesting that the MyotonPRO is capable of detecting the variations in stiffness of MG, LG, AT, and PF at different ankle and knee positions.