AbstractThe aim was to verify the validity (i.e., study A) and reliability (i.e., study B) of the alternative maximal accumulated oxygen deficit determined using onlya supramaximal effort (MAOD_ALT)to estimate anaerobic capacity [i.e., estimated by the gold standard maximal accumulated oxygen deficit method (MAOD)] during cycling. In study A, the effects of supramaximal intensities on MAOD_ALT and the comparison with the MAOD were investigated in fourteen active subjects (26 ± 6 years). In study B, the test-retest reliability was investigated, where fourteen male amateur cyclists (29 ± 5 years) performed the MAOD_ALT twice at 115% of the intensity associated to maximal oxygen uptake ("Equation missing"). MAOD_ALT determined at 130 and 150% of "Equation missing" was lower than MAOD (p ≤ 0.048), but no differences between MAOD_ALT determined at 100, 105, 110, 115, 120 and 140% of "Equation missing" (3.58 ± 0.53L; 3.58 ± 0.59L; 3.53 ± 0.52L; 3.48 ± 0.72L; 3.52 ± 0.61L and 3.46 ± 0.69L, respectively) with MAOD (3.99 ± 0.64L). The MAOD_ALT determined from the intensities between 110 and 120% of "Equation missing" presented the better agreement and concordance with MAOD. In the test-retest, the MAOD_ALT was not different (p > 0.05), showed high reproducibility when expressed in absolute values (ICC = 0.96, p < 0.01), and a good level of agreement in the Bland-Altman plot analysis (mean differences ± CI95%:−0.16 ± 0.53L). Thus, the MAOD_ALT seems to be valid and reliable to assess anaerobic capacity in cycling.