High-frequency ultrasound of the skin has recently been introduced for assessment of systemic effects in the cutis and subcutis of oral and inhaled glucocorticoids in children. However, the use of high-frequency skin ultrasound in clinical trials is invalidated because important methodological aspects have not been addressed. The aim of the present study was to evaluate inter- and intraobserver, day to day and diurnal variations of measurement of thickness of cutis and subcutis, and the fraction of low echogenic pixels (fLEP) in the cutis and, furthermore, to assess effects of exercise on the cutis and subcutis and variations in subcutaneous thickness between anatomical locations in children with a high-frequency B-mode ultrasound scanning device.