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The aim of the study was to determine the between-trial and between-day reliability of the Glazer protocol and our multi-activity surface electromyography (sEMG) measurement protocol for pelvic floor muscle (PFM) evaluation. The bioelectrical activity of PFM was collected using an endovaginal electrode in 30 young, Caucasian, nulliparous women (age 22–27, 168.6 ± 5.1 cm, 57.1 ± 11.8 kg). The between-trial and between-day reliability of the original Glazer protocol and the new multi-activity sEMG protocol were assessed during the following phases: pre-baseline rest, phasic (flick) contractions, tonic contractions, endurance contraction, and post-baseline rest. The Glazer protocol was characterized by poor and moderate measurement reliability. The time-domain parameters for the rise and fall of the signal amplitude and median frequency showed poor between-trial and between-day reliability. The mean and peak amplitudes indicated mainly good between-trial and moderate between-days reliability. Our protocol showed moderate to excellent reliability of both time-domain and quantitative parameters of muscle recruitment. In our protocol, the frequency-domain parameters describing muscle fatigue demonstrated much higher reliability than in the case of the Glazer protocol. The most important information obtained in this study was the significant improvement of diagnostic validity in PFM bioelectrical activity evaluation. The higher reliability of our sEMG protocol compared to original Glazer protocol allowed us to suggest that protocol modifications and changes in sEMG signal processing methods were effective in the improvement of PFM assessment quality. The new parameters calculated from the sEMG signal proposed in our sEMG protocol allowed us to obtain additional clinically important information about PFM dysfunctions regarding specific deficits of muscle contraction such as decrease in muscle strength; endurance or coordination related to, e.g., stress urinary incontinence; or pelvic floor muscle imbalance after childbirth. Full article