Pro-inflammatory cytokines such as TNF-α and IL-1β are implicated in the pathogenesis of inflammatory diseases such as sepsis. In sepsis, myocardial depression is a leading cause of death. To design novel therapeutic approaches, it is essential that we understand how these cytokines mediate the cellular basis of this cardiac dysfunction. Previous studies, including Greensmith and Nirmalan (2013), demonstrate that perturbed intracellular calcium handling and contractility can account for many aspects of myocardial depression. However, many of these studies suffer important limitations in that (A) small mammal models were used and (B) it remains unclear how pro-inflammatory cytokines affect certain key aspects of cellular function. To address this, we re-characterised the effects of TNF-α and IL-1β on intracellular calcium handling using a large animal model then investigated their effects on the ryanodine receptor (RyR) and myofilament sensitivity to calcium. Sheep ventricular myocytes were used for all experiments. All procedures used accord with the Animals (Scientific Procedures) Act, UK, 1986 and Directive 2010/63/EU of the European Parliament. Intracellular calcium and contractility dynamics were measured by epi-fluorescent photometry and video sarcomere detection respectively. Cells were separately exposed to 50 ng/ml TNF-α and IL-1β acutely and when required, excited using field stimulation (0.5 Hz). Intracellular Ca2+ waves were facilitated using 0.3 mM Ouabain and 5 mM Ca2+. SR Ca2+ content was estimated using the amplitude of 10 mM caffeine-evoked Ca transients. TNF-α and IL-1β decreased SR Ca2+ content by (mean±SEM) 27±3 (p<0.05) and 41±3 (p<0.05) % respectively accounting for a 17±3 (p<0.05) and 24±7 (p<0.05) % decrease of systolic calcium. Only with TNF-α did reduced systolic Ca2+ translate to reduced systolic shortening (20±8 (p<0.05) %). We observed no negative effect on the rate of systolic or caffeine-evoked Ca2+ decay. TNF-α and IL-1β decreased Ca2+ wave amplitude by 46±6 (p<0.001) and 34±7 (p<0.001) % and increased frequency by 38±8 (p<0.001) and 28±11 (p<0.001) %. Both cytokines reduced SR Ca2+ threshold for waves. In saponin-permeabalised cells the degree of sarcomere shortening over equivalent calcium ranges was attenuated. These findings confirm that in sheep, TNF-α and IL-1β decrease SR Ca2+ content leading to reduced systolic Ca2+. The increase of Ca2+ wave frequency and decrease of amplitude suggests both cytokines increase ryanodine receptor open probability; confirmed by a decreased SR Ca2+ threshold for waves. So while the reduction of SR Ca content does not appear to be SERCA-dependent, increased RyR leak may contribute. The loss of contractile response to increased Ca2+ suggests that both cytokines reduce myofilament Ca2+ sensitivity. These findings advance our understanding of cytokine-mediated cardiac intracellular Ca2+ dysregulation and provide additional cellular substrates for myocardial depression in sepsis.