Abstract Background: Anterior knee pain (AKP), commonly termed patellofemoral pain syndrome, represents the most prevalent lower extremity complaint in college-aged populations, affecting approximately 25% of physically active students. While quadriceps muscle weakness has been extensively characterized in patellofemoral pain pathophysiology, the role of muscle stiffness—a distinct biomechanical property reflecting tissue resistance to deformation—remains inadequately explored. Previous investigations have emphasized clinical flexibility testing and muscle length assessment; however, objective quantification of intrinsic muscle stiffness and its mechanistic relationship to patellofemoral joint loading remains limited. Objective: This study aimed to evaluate the association between quadriceps muscle stiffness, assessed via non-invasive myotonometry, and patellofemoral joint reaction force (PFJRF), calculated through inverse dynamics biomechanical modeling, in college students with and without anterior knee pain. Secondary objectives included examining the discriminative validity of myotonometric measurements for identifying individuals with AKP and determining whether regional variations in quadriceps stiffness (rectus femoris, vastus medialis, vastus lateralis) differentially correlate with joint reaction forces and functional performance metrics. Methods: A case-control biomechanical study was conducted with 185 college students (aged 18-25 years; n=93 AKP, n=92 pain-free controls) recruited through stratified sampling from university athletics and general student populations. Quadriceps muscle stiffness was measured via myotonometry (MyotonPRO) at standardized anatomical sites on the dominant leg in supine positioning. Patellofemoral joint reaction forces were calculated through inverse dynamics analysis of recorded lower extremity movement during standardized functional tasks (static squat, step descent, lateral step-down). Anterior knee pain severity was quantified using the Anterior Knee Pain Scale (AKPS) and Visual Analog Scale (VAS). Pearson correlation analysis, independent-samples t-testing, and receiver operating characteristic (ROC) curve analysis were employed for statistical evaluation. Results: College students with anterior knee pain demonstrated significantly elevated quadriceps muscle stiffness compared with pain-free controls across all measured regions: rectus femoris (520±45 N/m versus 380±35 N/m, p<0.001), vastus medialis (485±52 N/m versus 365±38 N/m, p<0.001), vastus lateralis (510±48 N/m versus 375±40 N/m, p<0.001). A strong positive correlation was observed between overall quadriceps stiffness and PFJRF magnitude (r=0.94, p<0.001), indicating that each 100 N/m increase in mean stiffness corresponded to approximately 187 N (±32 N) elevation in peak PFJRF. Myotonometric stiffness measurements demonstrated excellent discriminative validity for identifying AKP status, with area under the receiver operating characteristic curve of 0.96 (95% CI: 0.93-0.98) and optimal cutoff values of 445 N/m (sensitivity 92.5%, specificity 94.6%). Regional analysis revealed vastus medialis stiffness as the strongest predictor of PFJRF variation among the three quadriceps components (r=0.91, p<0.001), accounting for 83% of explained variance. Pain severity demonstrated moderate positive correlation with quadriceps stiffness (r=0.68, p<0.001) and strong correlation with PFJRF (r=0.76, p<0.001). Functional testing revealed that students with elevated quadriceps stiffness exhibited reduced knee flexion range during controlled descent movements (mean 8.2° less knee flexion, p=0.003) and increased vertical ground reaction forces (mean 22% elevation, p<0.001) compared with compliant-muscle control participants. Conclusions: Elevated quadriceps muscle stiffness represents a potentially modifiable biomechanical risk factor in college-aged individuals with anterior knee pain, associated with substantial elevation in patellofemoral joint loading. The strong association between muscle stiffness and PFJRF suggests that stiffness reduction through targeted soft tissue interventions may constitute an evidence-based mechanism for reducing joint compressive forces and pain severity in this population. Myotonometric assessment of quadriceps stiffness offers clinicians an objective, portable, and reliable method for quantifying muscle biomechanical properties, identifying at risk individuals, and monitoring therapeutic response. Future research should examine whether stiffness-reduction interventions effectively mitigate joint loading and improve clinical outcomes in college athletes and recreationally active students.