Interpretation of ultrasonographical measurements requires an understanding of the source and the magnitude of variation. A substantial part of the variation can be attributed to the observer, the equipment or the animal. The aim of this study was to evaluate which adrenal gland measurement is the least variable within and between observers. Three experienced ultrasonographers examined six cats at three different times on the same day, more than 1 h apart, according to a strict scanning protocol. Seven ultrasonographical measurements were performed on each adrenal gland (maximal length on sagittal images, maximal height at the cranial and caudal poles on sagittal and transverse images, and maximal width of the cranial and caudal poles on transverse images). Height measurements in both planes showed the lowest variability within and between observers compared with length and width measurements. Descriptive ultrasonographical features, such as echogenicity of the gland, presence of hyperechoic spots or layering assessment, demonstrated satisfactory-to-good intra- and inter-observer agreement, whereas the shape assessment showed very poor inter-observer agreement. The results of this study describe a reliable scanning protocol that can be the basis for future adrenal ultrasonographical examinations for cats suspected of adrenal disease (eg, hyperaldosteronism, hyperadrenocorticism, sex hormone-producing tumours).