Los Machucos - Chris Froome's Vuelta a Espana lead was cut by 42 seconds to 1:16 over Vincenzo Nibali as Austrian Stefan Denifl claimed a stunning win on Wednesday's 17th stage.
Denifl attacked out of a breakaway with five kilometres to go to hold off a brave chase by Alberto Contador to win a brutal, mountainous 180.5km stage from Villadiego Los Machucos in 4hr 48min 52min.
Contador was 28 seconds back to aid his chances of making the general classification podium with Colombia's Miguel Angel Lopez 1:04 down in third.
Froome has looked assured in his bid to become just the third rider to win the Tour de France and Vuelta in the same year.
But he seemed to pay for his efforts in winning Tuesday's time trial as he cracked on the final climb and couldn't stay with Nibali.
The Italian finished alongside Lopez in fourth with Froome down in 14th.
"I don't think anyone enjoys gradients over 25 percent but that's just how it is and it's the same for all of us," said Froome.
The Briton has three more competitive stages to defend his lead before Sunday's traditional parade around Madrid with the queen stage up the Alto de l'Angliru still to come on Saturday.
"It's never nice to lose time but I feel good. Three more days and I'm confident we can get the job done," he added.
Denifl's win is by a distance the biggest in Irish team Aqua Blue Cycling's short history.
Aqua Blue needed an invite to take part in the race in their first year as a professional team and also had to overcome the misfortune of their team bus being hit by an arson attack earlier in the event.
Denifl was part of an early six-man breakaway and stayed out on in front of the general classification favourites despite immense pressure from Contador in the final stages.
The Spanish veteran didn't get the stage win he desired in his final race before retirement.
However, Contador, a three-time Vuelta winner, is now just 1:21 down on Wilco Kelderman in the battle for the podium as he remains fifth overall.
The terrain eases slightly for the 169km 18th stage from Suances to Santo Toribio de Liebana on Thursday.
Results of the Vuelta a Espana 17th stage, a 180.5km ride from Villadiego to Los Machucos on Wednesday:
1. Stefan Denifl (AUT/ABS) 4h48min 52sec, 2. Alberto Contador (ESP/TRE) at 0:28sec, 3. Miguel Ángel López (COL/AST) 1:04, 4. Vincenzo Nibali (ITA/BAH) 1:04, 5. Ilnur Zakarin (RUS/KAT) 1:04. 6. Rafal Majka (POL/BOR) 1:04, 7. Michael Woods (CAN/CAN) 1:13, 8. Daniel Moreno (ESP/MOV) 1:17, 9. Wilco Kelderman (NED/SUN) 1:19, 10. David De la Cruz (ESP/QST) 1:42, 11. Steven Kruijswijk (NED/LNL) 1:42, 12. Jack Haig (AUS/ORI) 1:42, 13. Mikel Nieve (ESP/SKY) 1:46, 14. Chris Froome (GBR/SKY) 1:46, 15. Wout Poels (NED/SKY) 1:53, 16. Julian Alaphilippe (FRA/QST) 2:01, 17. Fabio Aru (ITA/AST) 2:04, 18. Peio Bilbao (ESP/AST) 2:04, 19. Louis Meintjies (RSA/EAU) 2:05, 20. Romain Bardet (FRA/ALM) 2:14
22. Nicolas Roche (IRL/BMC) 2:32, 24. Tejay Van Garderen (USA/BMC) 2:55, 43. Esteban Chaves (COL/ORI) 5:52
1. Chris Froome (GBR/Sky) 67hr 44min 03sec, 2. Vincenzo Nibali (ITA/BAH) at 1:16sec, 3. Wilco Kelderman (NED/SUN) 2:13, 4. Ilnur Zakarin (RUS/KAT) 2:25, 5. Alberto Contador (ESP/TRE) 3:34, 6. Miguel Angel Lopez (COL/AST) 4:39, 7. Michael Woods (CAN/CAN) 6:33, 8. Wouter Poels (NED/SKY) 6:40, 9. Fabio Aru (ITA/AST) 6:45, 10. David De la Cruz (ESP/QST) 10:10, 11. Steven Kruijswijk (NED/LNL) 10:13, 12. Esteban Chaves (COL/ORI) 10:46, 13. Tejay Van Garderen (USA/BMC) 11:20, 14. Louis Meintjes (RSA/EAU) 13:03, 15. Mikel Nieve (ESP/SKY) 23:20