The role of testosterone (T) in the regulation of cardiovascular function in females is not well understood. Our goal was to examine the effect of T on cardiomyocyte biology by measuring sarcomere shortening/relaxation and intracellular calcium cycling in adult female Sprague-Dawley rats. The rats were divided into the following four groups: (1) sham operated; (2) ovariectomized (OVX); (3) OVX plus T; and (4) OVX + T plus an aromatase inhibitor (AI). The final group was added to rule out effects from bioconversion of T to oestradiol. Sarcomere/calcium dynamics were measured after 4 weeks at 2 and 6 Hz, then at 6 Hz following exposure to 300 nm isoprenaline. Additionally, the acute (i.e. non-genomic) effects of T were evaluated in sham-operated and OVX + T + AI rats. There were no group differences, nor was there evidence for an effect of T on frequency or isoprenaline response. Additionally, there were no findings to indicate an acute, non-genomic T effect. Moreover, the relative α- and β-myosin heavy chain isoform complement was unchanged by OVX or T replacement. Our results argue against acute or chronic effects of T on cardiomyocyte shortening dynamics, calcium cycling or myosin heavy chain expression, arguing against any direct effect of T on cardiomyocyte function in adult females.