Controlled laboratory study. To determine the 6 degrees of freedom of lumbar vertebra in vivo during different functional activities in young and middle-aged asymptomatic subjects. A total of 26 asymptomatic subjects (M/F, 15/11; age, 20-55 years) were recruited in this study. They were divided into two groups: young group (number: 14; age: 20-30 years old) and middle-aged group (number: 12; age: 45-55 years old). The lumbar segment of each subject was scanned by computed tomography for the construction of three-dimensional (3D) models of the vertebra from L1 to S1. The lumbar spine was imaged by using a dual fluoroscopic system when the subjects performed different trunk postures. The 3D models of vertebrae were matched to two fluoroscopic images simultaneously in software. The range of motion (ROM) of vertebrae in the young and middle-aged groups was compared by using multiway analysis of variance, respectively. During the supine to the upright posture, vertebral rotation of the L1-S1 occurred mainly around the mediolateral axis (mean: 3.9 ± 2.9°). Along the mediolateral axis, vertebral translation was significantly lower at L1-2 (7.7 ± 2.4 mm) and L2-3 (8.0 ± 3.5 mm) than at L3-4 (1.6 ± 1.2 mm), L4-5 (3.3 ± 2.6 mm), and L5-S1 (2.6 ± 1.9 mm). At the L4-5 level, the young group had a higher rotational ROM than the middle-aged group around all three axes during left-right bending. Along the anteroposterior axis, the young group had a lower translational ROM at L4-5 than the middle-aged group during left-right bending (4.6 ± 3.3 vs. 7.6 ± 4.8 mm; P < 0.05). At L5-S1, the young group had a lower translational ROM than the middle-aged group during flexion-extension, left-right bending, and left-right torsion. This study explored the lumbar vertebral ROM at L1-S1 during different functional postures in both young and middle-aged volunteers. There were higher coupled translations at L3-4 and L4-5 than at the upper lumbar segments during supine to upright. The vertebral rotation decreased with age. In addition, the older subjects had a higher anteroposterior translation at the L4-5 segment and higher mediolateral translation at the L5-S1 segment than the young group. These data might provide basic data to be compared with spinal pathology.