Mehmet Topal's sweetly-struck volley condemned a new-look Republic of Ireland side to friendly defeat in Turkey.
O'Neill handed senior debuts to Declan Rice, Scott Hogan and substitute Matt Doherty and first starts to Alan Browne and Sean Maguire in Antalya, but it was Fenerbahce midfielder Topal who ultimately won the day with a 52nd-minute strike to clinch a 1-0 win.
Ireland came close to scoring in the first half, but a heavy touch from debutant Scott Hogan past the Turkish goalkeeper left him with a tight angle to score, and he only managed to put the ball into the side-netting.
West Ham's Rice and Aston Villa's Hogan were named in a team which included just three of the men who started the second leg of their World Cup play-off against Denmark in November - Shane Duffy, Jeff Hendrick and James McClean.
Skipper Seamus Coleman pulled on the green shirt for the first time since suffering a double leg fracture a day short of a year ago and there was a second cap for Bradford keeper Colin Doyle 10 years and 304 days after his first.
Turkey boss Mircea Lucescu also fielded a much-changed side, but retained Coleman's Everton team-mate Cenk Tosun in his starting line-up.
Hogan might have marked his debut with a goal against the run of play with just 18 minutes gone when he ran on to Hendrick's through-ball and rounded the keeper.
However, from an ever-tightening angle, he could only drag his shot into the side-netting.
Doyle had to make his first save of the game 10 minutes later when Tosun stepped outside central defender Shane Duffy and unleashed a fierce left-foot shot which the keeper parried before Kevin Long hacked clear.
Duffy heaved a sigh of relieved seconds later after he got his head to Gokhan Gonul's driven cross and almost directed it into his own goal.
The half became increasingly scrappy as it drew to a close, with the Turks on top but unable to make it count as the sides headed for their respective dressing rooms with the deadlock unbroken.
The home side finally forced their way ahead seven minutes after the restart when Yusuf Yazici crossed from Hakan Calhanoglu's short corner and Topal volleyed past the helpless Doyle.
Coleman's comeback ended after 62 minutes when he and Maguire were replaced by debutant Matt Doherty and Shane Long.
With the Republic trialling a 3-5-2 system, they were largely overrun in central midfield to leave Hogan, Maguire and later Long starved of meaningful possession.
However, there were positives with Rice turning in an assured performance on the left side of the defensive trio and later in midfield, and skipper Seamus Coleman getting 62 minutes under his belt a day short of a year since he suffered a horrific double leg fracture on international duty.