The objectives of this study were to evaluate the effect of supplemental premilking stimulation, provided after manual stimulation, by means of high-frequency pulsation without reduction of the vacuum in the pulsation chamber on milking performance, teat tissue condition, udder health, and well-being in dairy cows. In a randomized controlled trial, Holstein cows (n = 491) from 1 commercial dairy farm with a thrice-per-day milking schedule were assigned to treatment and control groups over a 64-d period. Treatments consisted of a maximum of 20 s of pulsation stimulation at a pulsation rate of 100 (SPS100) or 300 (SPS300) cycles per minute and a pulsation ratio of 25:75, which were applied after completion of manual premilking stimulation upon milking unit attachment. Cows in the control group (CON) received only traditional premilking stimulation by manual forestripping for 6 s, and wiping. Milking characteristics were measured with on-farm milk flow meters. Milking machine-induced short-term (swelling at teat base, firmness at teat end, and teat discoloration) and long-term (teat-end callosity) changes to the teat tissue were assessed manually and visually. Composite milk samples were analyzed for somatic cell counts. Cow hind-leg activity was assessed with 3-dimensional accelerometers. Fecal 11,17-dioxoandrostanes (11,17-DOAs; a group of cortisol metabolites) were determined on wk 4 and 8 to assess the well-being of the cows. Generalized linear mixed models were used to study the effect of treatments on the outcome variables milk yield/milking and milking unit-on time. We observed no meaningful differences among groups for milk yield or milking unit-on time. Least squares means and their 95% confidence intervals (95% CI) for cows in the SPS100, SPS300, and CON groups were 13.9 (13.3-14.5), 14.0 (13.4-14.6), and 13.9 (13.3-14.6) kg for milk yield and 218 (212-224), 218 (211-224), and 218 (212-224) s for milking unit-on time, respectively. Compared with cows in the CON group, the odds (95% CI) of short-term changes were 1.30 (0.95-1.78) for the SPS100 group and 1.50 (1.10-2.05) for the SPS300 group. The odds of long-term changes were 0.94 (0.67-1.34) for cows in the SPS100 group and 0.71 (0.49-1.04) for cows in the SPS300 group. We observed no differences in SCC. In reference to the CON group, the hazard ratio (95% CI) in SPS100 and SPS300, respectively, were 0.35 (0.13-0.98) and 1.22 (0.57-2.64) for clinical mastitis, and 0.34 (0.12-0.95) and 1.28 (0.60-2.73) for culling. The least squares means (95% CI) of hind-leg activity during milking were 8.3 (6.5-10.5), 10.6 (8.1-13.7), and 9.1 (7.2-11.6) movements per milking for the SPS100, SPS300, and CON groups, respectively. The least squares means (95% CI) of fecal 11,17-DOAs concentration (ng/g) at the first and second test days, respectively, were 31.1 (28.1-34.2) and 22.3 (19.2-25.4) for the SPS100 group, 26.4 (23.4-29.4) and 25.2 (22.0-28.4) for the SPS300 group, and 24.8 (21.8-27.9) and 25.0 (21.7-28.3) for the CON group. We conclude that applying supplemental stimulation after manual stimulation through the high-frequency pulsation system tested here did not impart additional benefits to the milk harvesting process, teat tissue condition, somatic cell count, or the well-being of the animals.