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Patellofemoral pain syndrome (PFPS), the most common source of knee pain among physically active individuals, is defined as anterior knee pain involving the patella and retinaculum that excludes other intraarticular and peri-patellar pathology. Differences in the activation and endurance of the quadriceps components, causing an imbalance in the forces controlling patellar tracking during flexion and extension in the knee joint, appear to be the most commonly reported risk factors for PFPS. The aim of this study was to determine the differences in bioelectric activity of vastus medialis and rectus femoris muscles during half knee bend position in runners with PFPS. The sample comprised 20 recreational runners (15 males, 5 females; aged 27 ± 5 years) reporting knee pain and diagnosed with PFPS. The age- and sex-matched control group consisted of recreational runners who did not report knee pain. The myon electromyographic system was used to determine the changes in the electromyographic median frequency during a half squat position. The decrease in the median frequency of vastus medialis and rectus femoris muscles was significantly greater in runners with PFPS in comparison to the control group (p < 0.01 for right and left vastus medialis and right rectus femoris; p < 0.05 for left rectus femoris). There is a relationship between knee pain related to PFPS and the imbalance of bioelectric activity of vastus medialis and rectus femoris muscles. Achieving the muscular balance within the thigh muscles can be an important factor in the rehabilitation process of PFPS.