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The aim of this study was to validate in vivo measurement of intravascular ultrasound (IVUS) for the analysis of structural and functional vessel wall alterations in a chronic animal model. Furthermore, the relation between functional and structural alteration of the vessel wall should be investigated. Fifteen cholesterol-fed rabbits (1%) and 15 control rabbits underwent balloon injury of the abdominal aorta. Immediately before and after balloon traumatization as well as 2 and 6 weeks later IVUS depiction of 10 aortal vessel segments was performed (n = 1100 measurements). In vivo IVUS measurements and morphometric analysis of the neointimal area of same aortal segments showed a high correlation (n = 148, r = 0.844, p < 0.001). Plaque area determined by morphometry revealed larger areas than the evaluation by IVUS (0.162 ± 0.138 vs. 0.130 ± 0.126 mm2, p < 0.001). Before balloon traumatization, pulsatility of the aortal vessel segments was less in cholesterol-fed rabbits (0.067 vs. 0.090, p < 0.01) and neointimal index higher (0.003 vs. 0). Investigation using IVUS 2 and 6 weeks after balloon traumatization demonstrated a continuous loss of arterial distensibility and an increase of neointimal index, being more pronounced in the cholesterol-fed group. As demonstrated by IVUS the loss of distensibility preceded the atherosclerotic alterations. Our investigation suggests using IVUS in this animal model is a reliable setting for long-term investigation of characteristics of the vessel wall. We could demonstrate that altered function of the vessel wall precedes the structural atherosclerotic vessel wall alterations.