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Overload training is hypothesized to alter autonomic regulation, although interpretations using indirect measures of heart rate variability are conflicting. The aim of the present study was to examine the effects of overload training on muscle sympathetic nerve activity (MSNA), a direct measure of central sympathetic outflow, in recreational endurance athletes.Measurements of heart rate variability, cardiac baroreflex sensitivity (BRS), MSNA (microneurography), and sympathetic BRS were obtained in 17 healthy triathletes and cyclists after 1 wk of reduced training (baseline) and again after 3 wk of either regular (n = 7) or overload (n = 10) training.After training, the changes (Δ) in peak power output (10 ± 10 vs −12 ± 9 W, P < 0.001), maximal heart rate (−2 ± 4 vs −8 ± 3 bpm, P = 0.006), heart rate variability (SD of normal-to-normal intervals, 27 ± 31 vs −3 ± 25 ms; P = 0.04), and cardiac BRS (7 ± 6 vs −2 ± 8 ms·mm Hg−1, P = 0.02) differed between the control and overload groups. The change in MSNA burst frequency (−2 ± 2 vs 4 ± 5 bursts per minute, P = 0.02) differed between groups. Across all participants, the changes in resting MSNA and peak power output were correlated negatively (r = −0.51, P = 0.04). No between-group differences in resting heart rate or blood pressure were observed (all P > 0.05).Overload training increased MSNA and attenuated increases in cardiac BRS and heart rate variability observed with regular training. These results support neural adaptations after overload training and suggest that increased central sympathetic outflow may be linked with decreased exercise performance.