The Liverpool forward played 79 minutes during the clash in Braga and was replaced by PSG midfielder Vitinha.
Fernando Santos' side only needed a draw against their neighbors to secure a place in the Nations League finals next summer.
But a late winner from Alvaro Morata gave Spain the three points and denied Portugal a chance to claim their second victory in the competition. But despite his team falling to defeat, Jota was impressive on the left-flank.
The 25-year-old has only made one Premier League appearance this term due to relentless injury issues. Jota scored 25 times in 55 appearances for the Reds last term and that workload appears to have caught up with him.
But Jurgen Klopp will be hoping his forward will take confidence from his impressive display and play a big part in reviving Liverpool's season.
The competition is fierce to break into Portugal's attack, but Jota will have done his chances of reaching the upcoming World Cup no harm at all. The winger injected some much-needed pace into the team, although he does need to improve on his record of two goals in his previous nine appearances.
Following the defeat in Braga, supporters took to Twitter to heap praise on the forward for his energetic display:
Portugal forward admits 'it's not easy' after missing most pre-season
Jota missed most of Liverpool's preseason due to a hamstring injury, but he ended his long goalless run with a goal against the Czech Republic last week.
Speaking to Liverpool's official website, the Portugal star admitted that he is having a hard time getting up to speed with the game, as he stated:
"Yeah, not easy! Especially for me to not have a pre-season. Coming back from injury [you] always wish for a different scenario and everything is rolling and we are winning the games comfortably but it is what it is. We need to think and to deal with the situation and I think I did that, and the team did that, so all good."
He further added:
"It was the first time in my career that happened but we cannot change that. I think I need to do my best from now on. Obviously [the] coach knows that as well. I’ve been playing gradually more and more so I can be ready to play 90 minutes soon."
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