The aims of the present study were to evaluate test-retest intra-observer repeatability of ultrasound measurement of the morphology and function of the pelvic floor muscles (PFMs). Seventeen subjects were tested twice. Two-, three- and four- dimensional ultrasound recorded cough, huff, muscle morphology and PFM contraction, respectively. Analyses were conducted offline. Measurements of levator hiatal dimensions demonstrated intra-class correlation coefficient (ICC) values of 0.61, 0.72, 0.86 and 0.92, for the anterior-posterior dimension, transverse dimension, resting area and narrowing during contraction, respectively. Muscle thickness showed variable reliability. ICC values for measurement of the position of the bladder neck were 0.86 and 0.82 at rest, in the vertical and horizontal direction. Displacement of the bladder neck during contraction, huff and cough demonstrated ICC values of 0.56, 0.59 and 0.51, respectively. Perineal ultrasound is a reliable method for measuring most of the tested parameters of morphology and function of the PFMs.