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Voluntary anal contraction plays an important role in fecal continence. Development ofprotocols able to assess the functional network involved is needed to characterize its neurobiological basis. We aimed topresent a novelprotocol based on functional magnetic resonance imaging (fMRI) and simultaneous anal manometry. Manometry recordings were used to build subject-optimized fMRI analyses which mapped brain regions involved in anal contraction.In 12 healthy women (45-61 years), a series of 120 whole-brain EPI-BOLD MRI images was obtained. During imaging, subjects were cued to perform anal contractions, registered by an anal manometer. Image analysis was done using SPM8. Individualized General Linear Models (GLM) were built using the anal manometry recordings. Fitting the GLM to the fMRI images identified the brain structures coupled with contractions.Anal manometry recordings revealed that all subjects followed accurately the contraction cues. Group manometry-based fMRI analysis revealed significant activations in medialprimary motor cortices (peak activation T value = 10.5; P < 0.000001), bilateral insula (T = 9.6; P < 0.000001), supplementary motor area (T = 8.4; P < 0.000002), bilateral putamen (T = 7.9; P < 0.000004) and cerebellum (T = 11.8; P < 0.000001).Theprotocol has been able to map the brain regions linked to voluntary anal sphincter contraction in healthy subjects.