Although the accumulation of intracellular calcium ions ([Ca²⁺]_i) is associated with muscle damage, little is known regarding the temporal profile of muscle [Ca²⁺]_i under in vivo conditions and specifically the effects of different contraction types (e.g., isometric, ISO; eccentric, ECC) on [Ca²⁺]_i remain to be determined. The following hypotheses were tested: 1) For 90 min at rest an in vivo vs. in vitro preparation would better maintain initial [Ca²⁺]_i. 2) Compared with ISO, ECC contractions (50 times, 10 sets, 5 min interval) would lead to a greater increase of [Ca²⁺]_i. 3) Elevated [Ca²⁺]_i during ECC would be reduced or prevented by the stretch-activated ion channels (SAC) blocker streptomycin and gadolinium (Gd³⁺). Spinotrapezius muscles of Wistar rats were exteriorized (in vivo) or excised (in vitro). [Ca²⁺]_i was evaluated by loading the muscle with Fura2-AM and using a fluorescence imaging. [Ca²⁺]_i rose progressively beyond 40 min at rest under in vitro but not in vivo conditions during the 90 min. protocol. In vivo [Ca²⁺]_i increased more rapidly during ECC (first set) than ISO (fifth set) (P 0.05, vs. pre-contraction values). The peak level of [Ca²⁺]_i was increased by 21.5% (ISO) and 42.8% (ECC) after 10 sets (both P 0.01). Streptomycin and Gd³⁺ abolished the majority of [Ca²⁺]_i increase during ECC (69% and 86% reduction, respectively. P 0.01 from peak [Ca²⁺]_i). In conclusion, in vivo quantitative analyses demonstrated that ECC contractions elevate [Ca²⁺]_i significantly more than ISO contractions and that stretch-activated channels may play a permissive role in this response.