Calorie restriction (CR) is a dietary intervention known to improve health and lifespan in mice. Typically, mice undergoing CR are fed a restricted aliquot of food once per day, which is eaten rapidly and results in a prolonged fasting period thereafter. A recent study concluded that the fasting period is more important than restricted calorie intake, because when CR was achieved by dietary dilution under ad libitum feeding (CR-D) this did not recapitulate all beneficial effects (e.g. lifespan extension [1]). To examine this further and whether biological sex has an effect, we investigated how CR, with or without fasting, alters skeletal muscle over the lifespan in C57BL6 mice that were sampled at 1, 6, 12 or 18 months or followed until natural end of life. Mice were 20% calorie restricted, either with fasting (CR-T), or through being fed a cellulose-diluted diet ad libitum (CR-D). Controls were fed an undiluted diet ad libitum. Body weight and fat mass increased with age, lean mass and grip strength decreased, while CR by either method was able to slow these changes. Mice fed either of the CR regimes had increased lifespan relative to controls. Preliminary proteomic analysis showed that 243/1529 muscle proteins were altered between the two CR groups. Surprisingly, the highest up-regulated proteins in CR-T vs CR-D were in the de novo lipogenic pathway, which is not normally active in muscle to a significant extent. There were main effects of biological sex, time and diet on the physiological and proteomic parameters, but few 3-way interactions. These data suggest that both CR-T and CR-D have beneficial effects on skeletal muscle health and longevity but may do so via different mechanisms.