Cytokines including tumour necrosis factor-alpha (TNF-α) are known to mediate systolic and diastolic myocardial dysfunction in systemic disease such as sepsis. To provide a cellular basis, previous work by Greensmith & Nirmalan (2013) studied the effects of 50 ng/ml TNF-α on cellular Ca handling and contractility in rat ventricular myocytes. TNF-α reduced sarcoplasmic reticulum (SR) Ca content, the amplitude of systolic Ca and cell shortening. TNF-α did not decrease SERCA activity so the mechanism underlying the reduced SR Ca remains unknown. In some cells, preceding a decrease, TNF-α produced a first beat increase in systolic Ca however, this was not investigated fully. The current study has two primary objectives. (1) to determine whether the fundamental observations previously reported are also true in a large mammal model and (2) investigate the apparent first beat increase of systolic Ca. All procedures used accord with the Animals (Scientific Procedures) Act, UK, 1986 and Directive 2010/63/EU of the European Parliament (Home Office, 1986). Ventricular myocytes were isolated from young (aged 18 months) sheep, and loaded with the ratiometric Ca indicator Fura-2. Cells were field stimulated at 0.5 Hz and intracellular Ca and contractility dynamics measured by epi-fluorescent photometry and video sarcomere detection respectively. Relative changes in SR Ca content were estimated from the amplitude of Ca transients evoked by application of 10 mM caffeine. 50 ng/ml TNF-α reduced SR Ca content by 27 %, accounting for a 17 and 20 % reduction in the amplitude of systolic Ca and sarcomere shortening respectively. The rate constant of systolic Ca decay was unaffected by TNF-α. In all cells the onset of systolic Ca decrease was rapid (<10 s). However, in 74 % of cells this was preceded by an immediate increase in systolic Ca (mean 58 %) and sarcomere shortening (mean 329 %) lasting for only one beat. Diastolic Ca was decreased by 4 % whilst resting sarcomere length was decreased by 0.5 %. With the exception of systolic sarcomere shorting, no effect was reversed upon washout. The decrease in SR Ca thence systolic Ca and contraction is in agreement with previous work confirming that in sheep alterations to Ca handling can provide a cellular basis for aspects of systolic dysfunction associated with sepsis. Here too, an effect on SERCA is not responsible for decreased SR Ca, however the first beat increase in systolic Ca does appear to be a real phenomenon suggesting that RyR potentiation may play a role – a mechanism we are currently investigating. In contrast to previous work, we observed a decrease in resting sarcomere length which could account for certain aspects of myocardial diastolic dysfunction. Interestingly however, diastolic Ca was also decreased, suggesting an effect on myofilament sensitivity may be responsible.