Multicellular cardiac muscles are widely used to study cardiac (patho-)physiology in vitro. One of the potential pitfalls of such experiments is that muscles with a large diameter have a larger diffusion barrier for transport of oxygen and waste products and can thus potentially form a hypoxic core. Although a sufficiently small muscle size is critical for obtaining unambiguous data, the relationship between muscle diameter and contractile performance specifically under near-physiological conditions remains unknown. Small uniform trabeculae of various diameters isolated from LBNF1 rats were stimulated at different temperatures (27.5-37.5 degrees C) and frequencies (1-8 Hz). Twitch contractions and rapid cooling contractures were used to assess contractile performance and SR Ca2+ load, respectively. We observed that at physiological frequencies and temperatures, contractile performance was clearly diminished in muscles with diameter >150 microm, likely due to the decreased rates of oxygen supply and waste removal. At room temperature slower contractions allow sufficient time for oxygen diffusion into the muscle core, and as a result the difference in contractile performance between the thin and thick muscles was less. Thus, in order to exclude adverse effects on contractile performance in multicellular myocardium under physiological conditions, it is essential that the preparations are of sufficient small diameter (<0.15 mm).