Mounting evidence suggests that the normal biomechanical state of arteries may include a nearly equibiaxial intramural stress, and that arteries tend to undergo rapid and dramatic remodeling when perturbed from this normal state. Technical developments since the early 1980s have enabled in vitro (acute) and ex vivo (chronic culture) study of isolated, perfused microvessels, and it is clear that these vessels share many functional similarities with arteries. To date, however, there has been no systematic study of the effects of in-plane biaxial loading on the biomechanical behavior of arterioles. Here, we describe a modification to a prior in vitro arterial test system that allowed us to investigate the role of altered axial stretch on the passive, myogenic, and norepinephrine-stimulated biaxial behavior of isolated rat cremaster arterioles. We show that axial stretches from 85% to 110% of values often used in the laboratory and consistent with those normally experienced in situ induce modest changes in the measured mean circumferential and axial stress-stretch behavior and in measures of distensibility and myogenic index. Nevertheless, altered axial stretch has a dramatic effect on the biaxial state of stress and nearly equibiaxial stresses occur at axial stretches larger than those typically used in isolated arteriole studies. This finding is consistent with estimates of material and functional behavior in arterioles and suggests that long-term ex vivo studies, wherein vessel growth and remodeling are critical, should be performed at higher axial lengths than have been used during most prior in vitro tests.