We evaluated age- and sex-dependent differences in heart rate variability (HRV) during the 6-min walk test (6MWT) in healthy adults. We also evaluated the intensity of the 6MWT based on HRV. 78 participants aged 40–49, 50–59, 60–69, and ≥70 years (42 females; 36 men) performed the 6MWT. Heart rate and HRV were monitored 1 min at rest and during the last 2-min of the test. The root mean square (RMSSD), instantaneous beat-to-beat variability (SD1), and long-term standard deviation (SD2) of RR intervals were calculated. The SD1 67% as the best cut-point for prediction of high-intensity exercise with 94% of sensitivity and 65% of specificity (area under the curve=0.804). We may conclude that autonomic modulation of heart rate during exercise was not dependent of age and sex. The HRV assessment during walking enables a valid estimation of exercise intensity in adults. We may therefore suggest the use of 6MWT for assessing exercise capacity and for prescribing exercises in adults aged 40 yrs and older.