Background: The investigation of soft tissue stiffness has garnered increasing interest due to its potential applications in detecting tissue conditions, monitoring therapy effects, and preventing sports injuries. This study utilized the MyotonPro as a reliable measurement device to assess muscle stiffness and muscle frequency in the vastus lateralis and medialis muscles of both the dominant and non-dominant legs. Methods: Sixteen young, healthy subjects (seven males and nine females, age 25 ± 3.46 years) participated in this study. To induce maximal muscle fatigue, the vastus lateralis and vastus medialis muscles were subjected to a 30 kg load using a single-leg leg press. Pre- and post-fatigue measurements were conducted by two testers on the dominant and non-dominant legs, respectively, employing the MyotonPro. Results: We revealed a significant increase in muscle stiffness after maximal muscle fatigue. Specifically, on the dominant side, the vastus lateralis exhibited a stiffness increase of 6.5%, while the vastus medialis showed a 6.3% increase. On the non-dominant side, the vastus lateralis demonstrated a 7.6% increase, and the vastus medialis exhibited a 6.7% increase in muscle stiffness. Furthermore, muscle frequency increased by 8.6% (vastus lateralis) and 13.5% (vastus medialis) on the dominant side and by 15.1% (vastus lateralis) and 6.3% (vastus medialis) on the non-dominant side. The reliability of the measurements varied, with Cronbach’s alpha values ranging from inadequate 0.49 to very good 0.88. Conclusion: This study affirms the efficacy of the MyotonPro as a measurement device for assessing muscle stiffness and establishes its reliability. The observed increase in muscle stiffness after maximal muscle fatigue, accompanied by changes in muscle frequency, underscores the device’s utility. However, further research is warranted to validate the reproducibility of these findings and explore additional facets of the muscular response to fatigue.