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Skeletal muscle fatigue is most often studied as a response to repeated stimulations in isometric conditions and it is usually quantified as the progressive loss of force generating capability over time. However, physical dynamic activity is based on the shortening of skeletal muscles. Therefore, the condition that best mimics body movements is the isotonic one, in which muscle is allowed to shorten against a constant load. In the literature, the isotonic fatigue test is performed allowing the muscle to lift a load corresponding to one-third of the maximal isometric force (reference optimal force), as best representative of the force at which the tissue develops its maximum power. The goal of this study was to devise a new testing protocol in which each muscle was tested for isotonic fatigue by shortening against its own optimal force, i.e. the force at which it really developed the maximum power. Our hypothesis was that testing all the muscle at a standard reference value would introduce significant errors in the parameters associated to muscle fatigue and in their variance. The proposed protocol was based on the real-time measurement of the maximum power a muscle was able to generate through the application of the after-load technique and a mathematical interpolation to the Hill's equation, that therefore allowed to determine the experimental optimal force to be applied during the fatigue test. Experimental results showed that the muscles tested with the experimental optimal force had a fatigue time significantly lower than the control muscles tested with the reference optimal force. A decrease, even if not statistically significant, was also measured for the power and work generated during the fatigue test. Of note, for all these parameters a huge decrease in the measurement variance was reported, confirming that a precise assessment of the muscle experimental optimal force was needed to increase the accuracy of the measurements. On the other hand, the application of the protocol proposed in this work required an increase in the test duration, due to the application of the after-load technique, and a real time measurement of the power generated by the tissue.