Purpose In vivo quantification of intervertebral motion through imaging has progressed to a point where biomarkers for low back pain are emerging. This makes possible deeper study of the condition's biometrics. However, the measurement of change over time involves error. The purpose of this prospective investigation is to determine the intrasubject repeatability of six in vivo intervertebral motion parameters using quantitative fluoroscopy. Methods Intrasubject reliability (ICC) and minimal detectable change (MDC) of baseline to 6-week follow-up measurements were calculated for six lumbar spine intervertebral motion parameters in 109 healthy volunteers. A standardised quantitative fluoroscopy (QF) protocol was used to provide measurements in the coronal and sagittal planes using both passive recumbent and active weight-bearing motion. Parameters were: intervertebral range of motion (IV-RoM), laxity, motion sharing inequality (MSI), motion sharing variability (MSV), flexion translation and anterior disc height change during flexion. Results The best overall intrasubject reliability (ICC) and agreement (MDC) were for disc height (ICC 0.89, MDC 43%) and IV-RoM (ICC 0.96, MDC 60%), and the worst for MSV (ICC 0.04, MDC 408%). Laxity, MSI and translation had acceptable reliability (most ICCs > 0.60), but not agreement (MDC > 85%). Conclusion Disc height and IV-RoM measurement using QF could be considered for randomised trials, while laxity, MSI and translation could be considered for moderators, correlates or mediators of patient-reported outcomes. MSV had both poor reliability and agreement over 6 weeks. Graphical abstract These slides can be retrieved under Electronic Supplementary Material.