OBJECTIVE To examine whether prolonged moderate exercising by untrained middle aged individuals of both sexes affects the plasma ratio of unsaturated to saturated (U/S) fatty acids in the non-esterified fatty acid (NEFA) and triacylglycerol (TG) fractions. METHODS Twenty two healthy untrained volunteers (eleven men and eleven pre-menopausal women), aged 35-55, exercised on bicycles at 50-55% maximal heart rate reserve (estimated 61% maximal oxygen uptake) for one hour. Pre- and post-exercise blood samples were analysed for lactate, glucose, glycerol, individual NEFAs and TG acyl groups, cholesterol, high density lipoprotein cholesterol, urea, cortisol, and testosterone. Biopsy specimens of adipose tissue were analysed for TG acyl group composition. RESULTS Serum total NEFAs increased significantly whereas total TG decreased significantly in both sexes. Changes in individual fatty acids, in both the NEFA and TG fractions, generally paralleled changes in the total pool but were not proportionate. As a result, U/S NEFAs increased in both sexes, although significantly only in women. The shift was in the direction of the composition of the major source of blood NEFAs--that is, adipose tissue TG. U/S acyl groups of TG also increased in both sexes, although significantly only in men. CONCLUSIONS Prolonged moderate exercise increases the U/S ratio of serum NEFAs and TG. This may add to the beneficial effects of exercise given the protective role of unsaturated fatty acids against coronary heart disease.