AbstractThis study investigated how contraction frequency impacts the mechano‐energetics of cardiac muscle performing mechanical work. Left‐ventricular trabeculae were isolated from rat hearts and mounted in our work‐loop calorimeter to assess their function at physiological temperature (37°C) across three stimulation frequencies, 2 Hz, 3.5 Hz and 5 Hz, in a randomised sequence. Each trabecula was subjected to two experimental protocols: work‐loop contractions under a range of afterloads and isometric contractions under a range of muscle lengths. Two contraction protocols allowed the partition of the various components of energy expenditure during cardiac contraction. By simultaneously measuring force–length work and heat output, mechanical efficiency was calculated over a range of afterloads to determine the peak value. Our findings revealed that force production, activation heat (energy associated with Ca2+ cycling) and cross‐bridge heat were unaffected by stimulation frequency. Trabeculae produced greater work output per twitch at 2 Hz and 3.5 Hz than at 5 Hz. Positive correlations among work output, shortening extent and mechanical efficiency were detected. From these findings it was concluded that the higher work output at lower frequencies is associated with greater extent of shortening, which correlates to greater mechanical efficiency. This study highlights the mechano‐energetic advantage of ventricular trabeculae in terms of increased work output and energy efficiency gained from operating at lower contraction frequencies, supporting the notion that heart rate reduction produces direct benefits on cardiac energetics.