The objective of this study was to validate and apply 2 different methods to record changes in teat tissue related to machine milking. Teat wall diameter was measured via B-mode ultrasound cross sectioning with a 7.5-MHz linear probe. Teat tissue thickness was measured using a cutimeter (spring-loaded caliper, spring constant 6.5 N/cm, 0.5 N at closed jaws). Both methods were applied at the teat barrel, 2 cm above the teat tip. In experiment 1, 24 teats from freshly slaughtered cows were used to perform ultrasound imaging (12 teats) or cutimeter measurements (12 teats) while the teat cisterns were filled with water to increase the intracisternal pressure from 0 to 30 kPa in steps of 1 kPa. Teat tissue thickness did not change at an intracisternal pressure from 0 to 10 kPa but increased with intracisternal pressure at levels >10 kPa. In contrast, teat wall diameter decreased with intracisternal pressure between 0 and 7 kPa but did not significantly change at a pressure ≥7 kPa up to 30 kPa. Significant Pearson correlation coefficients between intracisternal pressure and teat wall diameter were observed from 0 to 7 kPa (r = -0.38), and between intracisternal pressure and teat tissue thickness from 10 to 30 kPa (r = 0.45). In experiment 2, ultrasound and cutimeter measurements were performed in 12 lactating Holstein cows. Measurements before and during milking, immediately after cluster removal, with normal milking or with a 5-min overmilking, were performed and continued at 5-min intervals for 60 min and at 10-min intervals until 120 min. Additionally, with the 5-min overmilking treatment, measurements were continued at 60-min intervals up to 10 h after milking. Teat wall diameter decreased in response to milk ejection, followed by a continuous increase during the course of milking, with highest values after 5 min overmilking. Teat tissue thickness did not change during milking but was significantly increased after overmilking. Teat wall diameter and teat tissue thickness recovered to premilking levels within 35 min after normal milking and within 60 min after overmilking. Until 10 h after overmilking, the teat wall diameter decreased steadily, whereas teat tissue thickness was unfluctuating. In the physiologically relevant range of intramammary pressure, ultrasound measurements of the teat wall were affected by both intramammary pressure and mechanical forces, whereas cutimeter measurements were not affected by the intramammary pressure.