This study aims to investigate the influence of isocitrate dehydrogenase gene family (IDH) mutations, World Health Organization (WHO) grade, and mechanical preconditioning on glioma and adjacent brain elasticity through standard monotonic and repetitive atomic force microscope (AFM) nanoindentation. The elastic modulus was measured ex vivo on fresh tissue specimens acquired during craniotomy from the tumor and the peritumoral white matter of 16 diffuse glioma patients. Linear mixed-effects models examined the impact of tumor traits and preconditioning on tissue elasticity. Tissues from IDH-mutant cases were stiffer than those from IDH-wildtype ones among anaplastic astrocytoma patients ( = 0.0496) but of similar elasticity to IDH-wildtype cases for diffuse astrocytoma patients ( = 0.480). The tumor was found to be non-significantly softer than white matter in anaplastic astrocytomas ( = 0.070), but of similar elasticity to adjacent brain in diffuse astrocytomas ( = 0.492) and glioblastomas ( = 0.593). During repetitive indentation, both tumor ( = 0.002) and white matter ( = 0.003) showed initial stiffening followed by softening. Stiffening was fully reversed in white matter ( = 0.942) and partially reversed in tumor ( = 0.015). Tissue elasticity comprises a phenotypic characteristic closely related to glioma histopathology. Heterogeneity between patients should be further explored.