Caloric restriction (CR) mitigates many detrimental effects of aging and prolongs life span. CR has been suggested to increase mitochondrial biogenesis, thereby attenuating age-related declines in mitochondrial function, a concept that is challenged by recent studies. Here we show that lifelong CR in mice prevents age-related loss of mitochondrial oxidative capacity and efficiency, measured in isolated mitochondria and permeabilized muscle fibers. We find that these beneficial effects of CR occur without increasing mitochondrial abundance. Whole-genome expression profiling and large-scale proteomic surveys revealed expression patterns inconsistent with increased mitochondrial biogenesis, which is further supported by lower mitochondrial protein synthesis with CR. We find that CR decreases oxidant emission, increases antioxidant scavenging, and minimizes oxidative damage to DNA and protein. These results demonstrate that CR preserves mitochondrial function by protecting the integrity and function of existing cellular components rather than by increasing mitochondrial biogenesis.