The aim of this in vitro study was to investigate the impact of saliva on the abrasion of eroded enamel using two measuring methods. A total of 80 bovine enamel specimens from 20 bovine incisors were allocated to four experimental groups (n = 20 specimens per group). After baseline surface microhardness (SMH) measurements and profilometry all specimens were subjected to erosion (2 min, 1% citric acid, pH: 3.6, 37°C). SMH was determined again, and the depths of the Knoop indentations were calculated. Thereafter, specimens were incubated in human saliva (group 1 – no incubation/control, group 2 – 0.5 h, group 3 – 1 h, group 4 – 2 h) before toothbrush abrasion was performed. After final SMH measurements and profilometry, indentations were remeasured, and surface loss was calculated. SMH did not return to baseline values regardless of the length of saliva incubation. Further, an irreversible substance loss was observed for all specimens. With the indentation method, significantly (p < 0.05) more substance loss was found for controls (least square means ± standard error of 198 ± 19 nm) than for groups 2–4 (110 ± 10, 114 ± 11, and 105 ± 14 nm). Profilometric assessment showed significantly more substance loss for controls (122 ± 8 nm) than for group 4 (106 ± 5 nm). Intraclass correlation for interrater reliability between measurement methods was low (0.21, CI: 0.1–0.3), indicating poor agreement. Exposure of eroded enamel to saliva for up to 2 h could not re-establish the original SMH. The amount of measured substance loss depended on the measurement method applied.