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Abstract Opportunistic computed tomography (CT), which repurposes clinical CT scans, has the potential to aid in osteoporosis screening and diagnosis. To convert CT scans to bone-equivalent densities without a calibration phantom, internal (or phantomless) calibration methods have been developed, using referent tissues of known density within the body. A key limitation is that intravenous iodinated contrast enhancement, which is routinely used in clinical settings, impacts quantitative measurements derived from these phantomless calibration techniques. Therefore, this cross-sectional study examined the influence of contrast enhancement on internal reference tissues and vBMD measures derived using phantomless internal calibration. Hematuria protocol CT scans (N=194) were retrospectively analyzed. This protocol included unenhanced and two enhanced CT scans from the nephrographic (~90-second delay) and urographic (~10-minute delay) phases. The vertebrae were automatically identified and segmented with labels for the vertebral body and trabecular bone compartment in the third lumbar vertebra. Internal density calibration was performed using our previously established method with internal referent tissues (air, adipose, skeletal muscle, and cortical bone) to estimate the effective energy of each scan. A linear correction, derived based on training data from the nephrographic phase, was applied to test datasets from both the nephrographic and urographic phases. The nephrographic phase increased the vertebral body vBMD by 9.90 ± 4.28 mg/cm3 (5.52%, p < 0.0001) and the urographic phase by 2.33 ± 3.26 mg/cm3 (1.33%, p < 0.0001) compared to unenhanced scans. Applying the linear equation derived from the nephrographic training data reduced vBMD differences in the nephrographic test data but increased the variation in the urographic phase vBMD, demonstrating the need for phase-specific correction. This study demonstrates that vBMD measurements from contrast-enhanced CT are subject to significant error unless the interval between contrast administration and scan acquisition can be accounted for.