The aim of this study was to identify three-dimensional microstructural changes of trabecular bone with age and gender, using micro-computed tomography. Human trabecular bone from two disease groups, osteoporosis and osteoarthritis was analyzed. A prior analysis of the effects of some procedure variables on the micro-CT results was performed. Preliminary micro-CT scans were performed with three voxel resolutions and two acquisition conditions. On the reconstruction step, the image segmentation was performed with three different threshold values. Samples were collected from patients, with coxarthrosis (osteoarthritis) or fragility fracture (osteoporosis). The specimens of the coxarthrosis group include twenty females and fifteen males, while the fragility fracture group was composed by twenty three females and seven males. The mean age of the population was 69 ± 11 (females) and 67 ± 10 years (males), in the coxarthrosis group, while in the fragility fracture group was 81 ± 6 (females) and 78 ± 6 (males) years. The 30 μm voxel size provided lower percentage difference for the microarchitecture parameters. Acquisition conditions with 160 μA and 60 kV permit the evaluation of all the volume’s sample, with low average values of the coefficients of variation of the microstructural parameters. No statistically significant differences were found between the two diseases groups, neither between genders. However, with aging, there is a decrease of bone volume fraction, trabecular number and fractal dimension, and an increase of structural model index and trabecular separation, for both disease groups and genders. The parameters bone specific surface, trabecular thickness and degree of anisotropy have different behaviors with age, depending on the type of disease. While in coxarthrosis patients, trabecular thickness increases with age, in the fragility fracture group, there is a decrease of trabecular thickness with increasing age. Our findings indicate that disease, age and gender do not provide significant differences in trabecular microstructure. With aging, some parameters exhibit different trends which are possibly related to different mechanisms for different diseases.