During pre-strain process of 316L austenitic stainless steel, the increase in dislocation density and the appearance of mechanical twinning lead to the variation of mechanical behavior with pre-strain. In this study, the effect of pre-strain on strength parameter and deformation energy was discovered by small punch test. The results show that yield strength and saturated elastic energy increase with the increase in pre-strain. But fracture energy decreases with the increase in pre-strain, and maximum load remains invariable. Load method and energy method were compared to estimate mechanical strength of pre-strained material. It is found that, energy method without artificial factor and with physical meaning is more suitable to estimate yield strength than load method, while load method is more suitable to estimate ultimate tensile strength than energy method. Moreover, the effects of pre-strain and temperature were coupled in the energy method to estimate yield strength of pre-strained specimens at different temperatures.