Hydrogen peroxide (H2O2) and calcium ions (Ca2+) are functional regulators of skeletal muscle contraction and metabolism. Although H2O2 is one of the activators of the type-1 ryanodine receptor (RyR1) in the Ca2+ release channel, the interdependence between H2O2 and Ca2+ dynamics remains unclear. This study tested the following hypotheses using an in vivo model of mouse tibialis anterior (TA) skeletal muscle. 1. Under resting conditions, elevated cytosolic H2O2 concentration ([H2O2]cyto) leads to a concentration-dependent increase in cytosolic Ca2+concentration ([Ca2+]cyto) through its effect on RyR1. 2. In hypoxia (cardiac arrest) and muscle contractions (electrical stimulation), increased [H2O2]cyto induce Ca2+ accumulation. Cytosolic H2O2 (HyPer7) and Ca2+ (Fura-2) dynamics were resolved by TA bioimaging in C57BL/6J male mice under four conditions: Elevated exogenous H2O2, Cardiac arrest, Twitch and Tetanic contractions. Exogenous H2O2 (0.1-100mM) induced a concentration-dependent increase in [H2O2]cyto (+55%,0.1mM; +280%,100mM) and an increase in [Ca2+]cyto (+3%,1.0mM; +8%,10mM). This increase in [Ca2+]cyto was inhibited by pharmacological inhibition of RyR1 by dantrolene. Cardiac arrest-induced hypoxia increased [H2O2]cyto (+33%) and [Ca2+]cyto (+20%) 50min post-cardiac arrest. Compared to exogenous 1.0mM H2O2 condition, [H2O2]cyto after tetanic contractions rose less than one-tenth as much, while [Ca2+]cyto was 4.7-fold higher. In conclusion, substantial increases in [H2O2]cyto levels evoke only modest Ca2+ accumulation via their effect on the sarcoplasmic reticulum RyR1. On the other hand, contrary to hypoxia secondary to cardiac arrest, increases in [H2O2]cyto from contractions are small, indicating that H2O2 generation is unlikely to be a primary factor driving the significant Ca2+ accumulation after, especially tetanic, muscle contractions.