The effects of muscle contractions on the profile of post-contraction resting intracellular Ca²⁺ ([Ca²⁺]_i) accumulation in Type I diabetes are unclear. We tested the hypothesis that, following repeated muscle contractions, the rise in resting [Ca²⁺]_i evident in healthy rats would be increased in diabetes rats and that these changes would be associated with a decreased cytoplasmic Ca²⁺ buffering capacity. Adult male Wistar rats were divided into diabetic (DIA: Streptozocin i.p.) and healthy (CONT) groups. Four weeks later animals were anesthetized and spinotrapezius muscle contractions (10 sets of 50 contractions) were elicited by electrical stimulaton (100 Hz). Ca²⁺ imaging was achieved using Fura2-AM in the spinotrapezius muscle in vivo (i.e., circulation intact). The ratio (340/380 nm) was determined from fluorescence images following each contractions set, for estimation of [Ca²⁺]_i. Also, muscle Ca²⁺ buffering was studied in individual myocytes microinjected with 2 mM Ca²⁺ solution. After muscle contractions, resting [Ca²⁺]_i in DIA increased earlier and more rapidly than in CONT (P < 0.05). Peak [Ca²⁺]_i in response to the [Ca²⁺]_i injection was significantly higher in CONT (25.8%) than DIA (10.2%). Subsequently, CONT decreased rapidly to plateau 9-10% above baseline whereas DIA remained throughout the 60 s measurement window. No differences in SERCA (Ca²⁺ uptake) levels were evident between CONT and DIA, whereas RyR (Ca²⁺ release) level and mitochondrial enzyme activity was decreased in DIA (P < 0.05). In conclusion, diabetes impairs resting [Ca²⁺]_i homeostasis following muscle contractions. Markedly different responses to Ca²⁺ injection in DIA versus CONT suggest fundamentally deranged Ca²⁺ handling.