This study aimed to evaluate changes in muscle contractile properties during a training microcycle in semi-professional female football players and explore their relationship with training load variables. Nineteen players (age: 23.9 ± 3.9 years; body mass: 60.6 ± 6.9 kg; height: 164.5 ± 6.7 cm) underwent myotonometric assessments of the biceps femoris (BF) and rectus femoris (RF) before and after the following training sessions: MD1 (i.e., 1 day after the match), MD3, MD4, and MD5. Training loads were quantified for each session, revealing significant variations, with MD4 exhibiting the highest values for high-speed running distance, number of sprints, and accelerations. Notably, MD3 showed the highest perceived exertion (RPE), while MD5 recorded the lowest total distance run. Myotonometric assessments indicated significant differences in stiffness of the RF in MD3 and BF in MD5, as well as RF tone in MD5. The findings underscore a notable relationship between training load and myotometric variables, particularly in muscle stiffness and tone. These results emphasize the need for further research to clarify how training loads affect muscle properties in female athletes.