The aims of this study were to analyse the effect of different body positions on pelvic floor muscle (PFM) assessment using digital muscle testing, manometry and transabdominal ultrasound. In addition, subject acceptance of each testing position was recorded. Subjects were 20 women's health physiotherapists. The testing protocol included the best of three maximum voluntary contractions tested in each of four positions (crook lying, supine, sitting and standing). Significant differences in muscle strength and subject acceptance between positions were found with each tool, most often between lying and upright positions. Digital muscle testing and vaginal squeeze-pressure scores were highest in the lying position, and vaginal resting pressure and transabdominal ultrasound scores were highest in the standing position. Subjects preferred the lying positions for internal examinations. The clinical significance of these differences and the reasons for these variations require further investigation.