Venus Williams was unable to win back-to-back WTA Tour matches for the first time since 2019 after letting a lead of a set and a break slip in Cincinnati.
On the day Williams received a wildcard for a record-extending 24th US Open, the 43-year-old American lost 1-6 6-2 6-1 to world number 24 Zheng Qinwen.
Williams, ranked 533rd, led 6-1 2-0 before losing 11 games in a row.
China's Zheng, 20, served out victory and plays top seed Iga Swiatek in the Western and Southern Open last 16.
Williams will now continue her preparations for the US Open, where she will extend her own record of Open era singles appearances.