Stem cells are generally collected using flow cytometry, but this method is not applicable when the cell surface marker is not well determined. Satellite cells, which are skeletal muscle stem cells, have the ability to regenerate damaged muscles and are expected to be applicable for treatment of muscle degeneration. Although the transcription factor Pax7 is a known specific marker of satellite cells, it is not located on the cell surface and therefore flow cytometry is not directly applicable. In the present study, we turned our attention to the stress tolerance of adult stem cells, and we propose long-term trypsin incubation (LTT) as a novel approach to collecting satellite cells from mouse and human skeletal muscles. LTT led to a remarkable increase in the ratio of Pax7(+) cells that retain normal myogenic stem cell function. In particular, human Pax7(+) cells made up approximately 30% of primary cultured cells, whereas after LTT, the ratio of Pax7(+) cells increased up to ~80%, and the ratio of Pax7(+) and/or MyoD(+) myogenic cells increased to ~95%. Once transplanted, LTT-treated cells contributed to subsequent muscle regeneration following repetitive muscle damage without additional cell transplantation. The stress tolerance of Pax7(+) cells is related to heat shock protein 27 and aB-crystallin, members of the small heat shock protein family. This approach, based on the stress resistance of adult stem cells, is a safe and inexpensive method of efficiently collecting human satellite cells and may also be used for collecting other tissue stem cells whose surface marker is unknown.