Background: Surgical resection of tongue cancer may impair swallowing and speech. Knowledge of tongue muscle architecture affected by the resection could aid in patient counseling. Diffusion tensor imaging (DTI) enables reconstructions of muscle architecture in vivo. Reconstructing crossing fibers in the tongue requires a higher-order diffusion model. Purpose: To develop a clinically feasible diffusion imaging protocol, which facilitates both DTI and constrained spherical deconvolution (CSD) reconstructions of tongue muscle architecture in vivo. Study Type: Cross-sectional study. Subjects/Specimen: One ex vivo bovine tongue resected en bloc from mandible to hyoid bone. Ten healthy volunteers (mean age 25.5 years; range 21–34 years; four female). Field Strength/Sequence: Diffusion-weighted echo planar imaging at 3 T using a high-angular resolution diffusion imaging scheme acquired twice with opposing phase-encoding for B0-field inhomogeneity correction. The scan of the healthy volunteers was divided into four parts, in between which the volunteers were allowed to swallow, resulting in a total acquisition time of 10 minutes. Assessment: The ability of resolving crossing muscle fibers using CSD was determined on the bovine tongue specimen. A reproducible response function was estimated and the optimal peak threshold was determined for the in vivo tongue. The quality of tractography of the in vivo tongue was graded by three experts. Statistical Tests: The within-subject coefficient of variance was calculated for the response function. The qualitative results of the grading of DTI and CSD tractography were analyzed using a multilevel proportional odds model. Results: Fiber orientation distributions in the bovine tongue specimen showed that CSD was able to resolve crossing muscle fibers. The response function could be determined reproducibly in vivo. CSD tractography displayed significantly improved tractography compared with DTI tractography (P = 0.015). Data Conclusion: The 10-minute diffusion imaging protocol facilitates CSD fiber tracking with improved reconstructions of crossing tongue muscle fibers compared with DTI. Level of Evidence: 2. Technical Efficacy: Stage 1. J. Magn. Reson. Imaging 2019;50:96–105.