Scotland failed to follow up their heroics of reaching a first major tournament for 22 years as Steve Clarke's men were beaten 1-0 by Slovakia on Sunday, their first defeat in 10 games.
Jan Gregus's deflected strike on 32 minutes separated the meeting of two teams who qualified for Euro 2020 this week as a much-changed Scotland were left to rue not taking a series of chances.
A first defeat in 13 months for the Scots means they have to wait to secure top spot in Nations League Group B2.
That could still happen on Sunday if the Czech Republic fail to beat Israel or if Scotland are victorious when they travel to Israel on Wednesday.
Clarke made eight changes from the side that beat Serbia on penalties on Thursday to reach the European championships.
However, the visitors still enjoyed the better of the chances and will feel aggrieved not to have at least extended their unbeaten run.
Oli McBurnie scored one of Scotland's five successful penalties in midweek but the Sheffield United striker is still searching for his first international goal from open play and wasted two huge chances to give his side the lead.
Craig Gordon returned in goal for the first time in over two years but was helpless when Gregus's shot clipped Kenny McLean and flew into the bottom corner.
Stuart Armstrong was inches away from an equaliser just before half-time as he flicked Ryan Christie's excellent through ball wide.
After the break it was Slovak goalkeeper Marek Rodak who ensured his side took all three points.
First the Fulham stopper made an incredible save to deny McLean's header from Kieran Tierney's cross.
Then with the last action of the game, Leigh Griffiths thought he had earned a point with a powerful low shot that Rodak turned behind.
Scotland still top Group B2 of the Nations League with a four-point lead over the Czechs, who they will face in their opening game of the Euros.
Slovakia, who qualified for Euro 2020 with a playoff win over Northern Ireland on Thursday, remain bottom of the group with four points from five games.