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To evaluate the intrasurgical retinal architectural and macular hole (MH) geometric alterations that occur during surgical MH repair using intraoperative optical coherence tomography.A retrospective, multisurgeon, single-center, consecutive case series of 21 eyes undergoing surgical repair for MH with concurrent intraoperative optical coherence tomography using a custom microscope–mounted optical coherence tomography system was performed. All patients underwent surgical repair with pars plana vitrectomy, membrane peel, and gas tamponade. A novel three-dimensional segmentation algorithm was used for volumetric analysis of intrasurgical changes of MH geometry after surgical repair. Intraoperative optical coherence tomographic characteristics analyzed included MH volume, minimum diameter, base area, and hole height. Outer retinal architecture changes were analyzed both quantitatively and qualitatively.All 21 eyes were successfully imaged with intraoperative optical coherence tomography. Nineteen of 21 eyes had images of sufficient signal strength to allow for quantitative analysis. Significant changes were noted in MH geometry after internal limiting membrane peeling including increased MH volume, increased base area, and decreased top area (all P < 0.03). Additionally, increased subretinal hyporeflectance was noted by expansion of the height between the inner segment/outer segment and retinal pigment epithelium bands (P = 0.008). Peeling methods and surgeon experience did not correlate with the magnitude of architectural alterations. Macular hole algorithm measurements and alterations were associated with visual outcome and MH closure.Significant alterations occur in MH geometry and outer retinal structure after internal limiting membrane peeling. These changes are subclinical and unable to be appreciated with en face surgical microscope viewing and require intraoperative optical coherence tomography for visualization. Preliminary analysis of these measurements identified an association with visual outcome and successful MH closure. The functional significance of these changes deserves further study.