The time necessary to obtain a steady state for an accurate and reliable assessment of resting [Formula: see text] remains unclear and was the purpose of this study. Thirty healthy men, aged 17-28 years, visited the laboratory twice for the assessment of resting [Formula: see text], which was assessed as follows: (a) 24 h abstention from physical exercise, alcohol, soft drinks and caffeine, (b) fasting for at least 8 h, (c) an acclimation period of 10 min, and (d) 60 min assessment in a supine position. Resting [Formula: see text] significantly changed during the 60 min (F = 37.4, P < 0.001), exhibiting a monoexponential decrease before reaching an asymptote. Post hoc pairwise comparisons showed that significant differences existed between consecutive means until the 30 min time point, after which there were no significant differences. The [Formula: see text] response across trials exhibited high test-retest reliability, with within-subject coefficients of variations at each time point ranging from 2.8 to 7.0 % and intraclass correlation coefficients ranging from 0.90 to 0.99. The reliability was higher from the 25 min time point onwards. Based on these findings, the following recommendations are made to promote accurate assessment of resting [Formula: see text]: (a) initiate the resting [Formula: see text] measurement with 10 min of acclimation to the assessment apparatus, (b) determine resting [Formula: see text] for a minimum of 30 min, until an apparent [Formula: see text] steady state has been achieved; and (c) determine resting [Formula: see text] for a further 5 min, with the average of this last 5 min of data being regarding as the resting [Formula: see text].