Background <p>Prolonged standing can induce discomfort and increase the risk of musculoskeletal disorders. The motion states of individuals who stand for prolonged periods can be categorized into stationary standing and intermittent movement. However, previous research predominantly focused on monitoring various indicators during stationary standing, neglecting intermittent movement. This missing may lead to an incomplete understanding of prolonged standing’s effects.</p> Methods <p>This study examined joint and muscle activity during both prolonged stationary standing and intermittent movement in 22 participants. Marker trajectory and electromyography data of all subjects were collected throughout the entire process, and their perceived discomfort was assessed through questionnaires. Paired t-tests were conducted to assess the effects of prolonged standing on joint kinematics, muscle fatigue and muscle synergy.</p> Results <p>The results demonstrated that prolonged standing affected participants’ lower limb joint kinematics, characterized by a reduced range of joint motion during stationary standing and increased hip external rotation during intermittent movement. The rectus femoris and vastus medialis exhibited fatigue, which was more pronounced during intermittent movement. Furthermore, muscle synergy analysis demonstrated that prolonged standing altered muscle coordination strategies. Participants with and without prolonged standing-related low back pain exhibited different trunk muscle reorganization patterns, yet both groups showed a shift in the center of muscle activity and a reduction in activation breadth.</p> Conclusion <p>The combination of stationary standing and intermittent movement provides more comprehensive insights than studying standing alone. These insights contribute to a deeper understanding of how prolonged standing affects joint kinematics and muscle activity.</p>