The effects of feeding a low-protein diet (5 percent w/w) and daily exercise on the rates of substrate (futile) cycling between triglyceride and fatty acids (TG-FA cycle) were studied in rats in vivo using a radiochemical assay that involves following the incorporation of tritium from 3H-H2O into the fatty acid and glycerol moieties of triglyceride. Sixty-four rats were fed either a purified control diet (COND) consisting of 70 percent carbohydrate, 20 percent protein, 5 percent fat or an experimental low protein (LPD) diet consisting of 80 percent carbohydrate, 5 percent protein and 10 percent fat (w/w) and were either exercised six days weekly or remained sedentary for six weeks. Both LPD and exercise training (EXT) were found to increase significantly the rate of TG-FA substrate cycling above the rates observed in dietary and sedentary control groups. The LPD increased significantly the rate of cycling in interscapular brown adipose (IBAT), while adipose (WAT) and diaphragm muscle. EXT increased the rates of substrate cycling in soleus, heart, and diaphragm muscle and WAT. Rate of cycling in cardiac or skeletal muscle was one-twentieth that found in adipose tissue. There were also sex differences in the rate of substrate cycling. Substrate cycling in soleus and heart muscle of male animals were consistently higher than respective female treatment groups. Sedentary and EXT LPD animals weighed significantly less than but consumed a similar amount of food to the respective COND animals. These data provide the first in vivo evidence that the rate of substrate cycling can be increased by diet or by exercise training. The possible importance of TG-FA and other substrate cycles on efficiency of energy metabolism and etiology of obesity are discussed.