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To test the utility of HR variability (HRV) in daily exercise prescription in moderately active (approximately two exercises per week) men and women.A total of 21 men and 32 women were divided into standard training (ST: males = 7 and females = 7), HRV-guided training (HRV-I: males = 7 and females = 7; HRV-II: females = 10), and control (males = 7 and females = 8) groups. The 8-wk aerobic training period included 40-min exercises at moderate and vigorous intensities (70% and 85% of maximal HR). The ST group was instructed to perform two or more sessions at moderate and three or more sessions at vigorous intensity weekly. HRV-I and HRV-II groups trained on the basis of changes in HRV, measured every morning. In the HRV-I group, an increase or no change in HRV resulted in vigorous-intensity training on that day. Moderate-intensity exercise or rest was prescribed if HRV had decreased. The HRV-II group performed a vigorous-intensity exercise only when HRV had increased. Peak oxygen consumption (V˙O2peak) and maximal workload (Loadmax) were measured by a maximal bicycle ergometer test before and after the intervention.The changes in V˙O2peak did not differ between the training groups either in men or in women. In men, the change in Loadmax was higher in the HRV-I group than in the ST group (30 ± 8 vs 18 ± 10 W, P = 0.033). In women, no differences were found in the changes in Loadmax between the training groups (18 ± 10, 15 ± 11, and 18 ± 5 W for ST, HRV-I, and HRV-II, respectively). The HRV-II group performed fewer vigorous-intensity exercises than the ST and HRV-I groups (1.8 ± 0.3 vs 2.8 ± 0.6 and 3.3 ± 0.2 times per week, respectively, P < 0.01 for both).HRV measurements are beneficial in exercise training prescription in moderately active men and women. Women benefit from HRV guidance by achieving significant improvement in fitness with a lower training load.