Virtual non-calcium (VNCa) images from dual-energy computed tomography (DECT) have shown high potential to diagnose bone marrow disease of the spine, which is frequently disguised by dense trabecular bone on conventional CT. In this study, we aimed to define reference values for VNCa bone marrow images of the spine in a large-scale cohort of healthy individuals. DECT was performed after resection of a malignant skin tumor without evidence of metastatic disease. Image analysis was fully automated and did not require specific user interaction. The thoracolumbar spine was segmented by a pretrained convolutional neuronal network. Volumetric VNCa data of the spine’s bone marrow space were processed using the maximum, medium, and low calcium suppression indices. Histograms of VNCa attenuation were created for each exam and suppression setting. We included 500 exams of 168 individuals (88 female, patient age 61.0 ± 15.9). A total of 8298 vertebrae were segmented. The attenuation histograms’ overlap of two consecutive exams, as a measure for intraindividual consistency, yielded a median of 0.93 (IQR: 0.88–0.96). As our main result, we provide the age- and sex-specific bone marrow attenuation profiles of a large-scale cohort of individuals with healthy trabecular bone structure as a reference for future studies. We conclude that artificial-intelligence-supported, fully automated volumetric assessment is an intraindividually robust method to image the spine’s bone marrow using VNCa data from DECT.