Rainbow smelt, Osmerus mordax, experience a wide range of temperatures in their native habitat. In response to cold, smelt express anti-freeze proteins and the osmolytes glycerol, trimethylamine N-oxide (TMAO) and urea to avoid freezing. The physiological influences of these osmolytes are not well understood. Urea destabilizes proteins, while TMAO counteracts the proteindestabilizing forces of urea. The influence of glycerol on muscle function has not been explored. We examined the effects of urea, glycerol and TMAO through muscle mechanics experiments with treatments of the three osmolytes at physiological concentrations. Experiments were carried out at 10°C. The contractile properties of fast-twitch muscle bundles were determined in physiological saline and in the presence of 50 mmol l −1 urea, 50 mmol l −1 TMAO and/or 200 mmol l −1 glycerol in saline. Muscle exposed to urea and glycerol produced less force and displayed slower contractile properties. However, treatment with TMAO led to higher force and faster relaxation by muscle bundles. TMAO increased power production during cyclical activity, while urea and glycerol led to reduced oscillatory power output. When muscle bundles were exposed to a combination of the three osmolytes, they displayed little change in contraction kinetics relative to control, although power output under lower oscillatory conditions was enhanced while maximum power output was reduced. The results suggest that maintenance of muscle function in winter smelt requires a balanced combination of urea, glycerol and TMAO.