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Background: 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., Methods: Three studies were conducted, each including 10 healthy prepubertal children. High-frequency skin ultrasound was performed with the 20 MHz Dermascan C (Cortex Technology, Hadsund, Denmark). In study 1, the same observer performed five consecutive scannings to assess intraobserver variations. In study two different observers performed scannings at 2 h intervals between 08:00 and 20:00 h, whereby interobserver and diurnal variations were assessed. In study 3, the same observer performed scannings in different anatomical locations on five consecutive days, and on one of these days before and after exercise. Thus day-to-day variations and the effect of exercise were assessed., Results: Low inter- and intraobserver variations were found on assessment of the thickness of cutis and subcutis, whereas high variations were found on evaluation of the dermal water content. Diurnal variations were absent, and day-to-day variations were low. Exercise caused significant increases in the thickness of cutis and subcutis on the thigh., Conclusion: Low inter- and intraobserver variations make high-frequency ultrasound a precise and reliable tool for assessment of the cutaneous and subcutaneous thickness in children. In future trials, repetitive scannings need not to be performed at the same time of the day, whereas strenuous physical activity should be avoided on days of examination.