Preclinical data acquired for human muscle stem (hMuStem) cells indicate their great repair capacity in the context of muscle injury. However, their clinical potential is limited by their moderate ability to survive after transplantation. To overcome these limitations, their encapsulation within protective environment would be beneficial. In this study, tunable calcium-alginate hydrogels obtained through molding method using external or internal gelation were investigated as a new strategy for hMuStem cell encapsulation. The mechanical properties of these hydrogels were characterized in their fully hydrated state by compression experiments using Atomic Force Microscopy. Measured elastic moduli strongly depended on the gelation mode and calcium/alginate concentrations. Values ranged from 1 to 12.5 kPa and 3.9 to 25 kPa were obtained for hydrogels prepared following internal and external gelation, respectively. Also, differences in mechanical properties of hydrogels resulted from their internal organization, with an isotropic structure for internal gelation, while external mode led to anisotropic one. It was further shown that viability, morphological and myogenic differentiation characteristics of hMuStem cells incorporated within alginate hydrogels were preserved after their release. These results highlight that hMuStem cells encapsulated in calcium-alginate hydrogels maintain their functionality, thus allowing to develop muscle regeneration protocols to improve their therapeutic efficacy.