The Williams sisters, a wild-card entry into the event, were beaten 2-6 6-1 10-8 by Ukrainian Marta Kostyuk and American Peyton Stearns.
The fan favourites bounced back from a rusty first set to dominate the next and force the 10-point tie-break, where the Williams sisters trailed 6-0 before rallying again to threaten a famous comeback.
- Latest scores and results from tennis' Cincinnati Open
- Latest Sky Sports deals or stream tennis with no contract
- Download Sky Sports app for analysis, news and video
Back-to-back double-faults by Kostyuk saw the Williams sisters reduce the deficit to 8-7, before the match ended anticlimactically with a double fault from Venus Williams.
"I thought it was fun," Serena said. "I thought it was special. We kicked off a little rust in the middle of the match and just, you know [were] starting to feel it.
"I didn't come out here to win this tournament. Winning is obviously great. But in this position just, you know, having fun. It was really cool to be able to do this again."
The Williams sisters, who together have won 14 Grand Slam doubles titles, were due to team up for doubles at Wimbledon but had to withdraw due to Serena's injury.
The sisters' appearance together was their first since the 2022 US Open and their first in a non-Grand Slam event since the Italian Open, with the match only Serena's fourth since returning to the WTA Tour this summer aged 44.
"Eventually we would find our way to each other," the 46-year-old Venus said about the decision to play doubles again. "It's our first time playing together in forever. It was great for us to get out there and do it."
Kostyuk and Stearns are playing in their first tournament together and will now face third seeds Guo Hanyu and Kristina Mladenovic in the next round.
"The crowd got the show," Kostyuk said. "They probably couldn't ask for anything better. At the end a tie-break is a lottery. You never know which way it's going to go."
Watch the ATP and WTA Tours live on Sky Sports. The US Open is also live on Sky Sports from August 30. Stream tennis with NOW.
