Objective: This study aimed to investigate the relationship between creatine kinase (CK) levels and the mechanical properties of lower limb muscles—specifically stiffness and elasticity—in order to propose a practical and time-efficient methodology for fatigue monitoring in professional athletes. Methods: Twenty-seven male professional soccer players participated in the study. Muscle stiffness and elasticity were measured using a handheld myotonometer (MyotonPro) at 23 reference points across the dominant lower limb. CK levels were assessed via venous blood samples collected 48 h post-match. Pearson correlation analysis was conducted to examine associations between CK and mechanical muscle properties. Results: Significant positive correlations were observed between CK levels and the stiffness of adductor longus (r = 0.57, p < 0.01), rectus femoris (r = 0.51, p < 0.05), and vastus medialis (r = 0.38, p = 0.05). A strong correlation was also found between CK and the elasticity of the biceps femoris (r = 0.56, p < 0.01). No significant relationships were detected for relaxation or creep parameters. These findings suggest that only a select group of CK-sensitive muscles may be sufficient for monitoring neuromuscular fatigue. Conclusion: While current fatigue monitoring practices often involve extensive muscle group assessments, this study proposes a simplified protocol based on the identification of fatigue-sensitive muscles with strong CK associations. By bridging the gap between biochemical and biomechanical evaluations, this approach offers a practical and scalable solution for fatigue monitoring in both clinical and sports performance settings.