Background context Prolonged sitting may alter the passive stiffness of the lumbar spine. Consequently, performing full lumbar flexion movements after extended periods of sitting may increase the risk of low back injury. Purpose The purpose was to quantify time-varying changes in the passive flexion stiffness of the lumbar spine with exposure to prolonged sitting and to link these changes to lumbar postures and trunk extensor muscle activation while sitting. A secondary objective was to determine whether men and women responded differently to prolonged sitting. Study design Passive lumbar flexion moment-angle curves were generated before, during and after 2 hours of sitting. Lumbar flexion/extension postures and extensor muscle activation levels were measured while sitting. Sample Twelve (6 men, 6 women) university students with no recent low back pain were studied. Outcome measures Quantified changes in the shapes of the passive flexion moment-angle curves (slopes, breakpoints and maximum lumbar flexion angles) were the outcome measures. While sitting, average lumbar flexion/extension angles, the distribution of lumbar flexion/extension postures, average electromyogram (EMG) amplitude, the number and average length of EMG gaps, and trunk extensor muscle rest levels were measured. Methods Participants performed deskwork for 2 hours while sitting on the seat pan of an office chair. Moment-angle relationships for the lumbar spine were derived by pulling participants through their full voluntary range of lumbar flexion on a customized frictionless table. Results Lumbar spine stiffness increased in men after only 1 hour of sitting, whereas the responses of women were variable over the 2-hour trial. Men appeared to compensate for this increase in stiffness by assuming less lumbar flexion in the second hour of sitting. Conclusions Changes in the passive flexion stiffness of the lumbar spine may increase the risk of low back injury after prolonged sitting and may contribute to low back pain in sitting.