Although pathologic changes to the structure and function of small blood vessels are hallmarks of various cardiovascular diseases, limitations of conventional investigation methods (i.e. pressure myography) have prohibited a comprehensive understanding of the underlying mechanisms. We developed a microfluidic device to facilitate assessment of resistance artery structure and function under physiological conditions (37 degrees C, 45 mmHg transmural pressure). The platform allows for on-chip fixation, long-term culture and fully automated acquisition of up to ten dose-response sequences of intact mouse mesenteric artery segments (diameter approximately 250 micrometres and length approximately 1.5 mm) in a well-defined microenvironment. Even abluminal application of phenylephrine or acetylcholine (homogeneous condition) yielded dose-response relationships virtually identical to conventional myography. Unilateral application of phenylephrine (heterogeneous condition) limited constriction to the drug-exposed side, suggesting a lack of circumferential communication. The microfluidic platform allows us to address new fundamental biological questions, replaces a manually demanding procedure with a scalable approach and may enable organ-based screens to be routinely performed during drug development.