Randy de Puniet set a surprise fastest time in the opening practice session at Assen, dislodging Valentino Rossi from the top spot in the final minute.
The LCR Honda sneaked ahead of the world champion by just 0.005 seconds when he produced a 1m37.842s with his penultimate lap of the afternoon.
Until then Yamaha had looked set to resume the form it showed at Barcelona by taking a one-two in practice, for Rossi and his team-mate Jorge Lorenzo had usurped long-time pace-setter Dani Pedrosa in the final quarter of an hour. Lorenzo was the first to beat the Honda, but he only stayed on top for a few moments before Rossi took charge.
Then with only a minute remaining de Puniet found the few tenths he required to move up from fifth to first.
Rossi remained second despite Casey Stoner (Ducati) beating his time around the first half of the lap. The Australian dropped speed in the remaining sectors and had to settle for third, with just 0.035s covering the top three.
Lorenzo ended up fourth, ahead of the factory Hondas, as Pedrosa slipped back to sixth behind team-mate Andrea Dovizioso.
Colin Edwards ran as high as second for Tech 3 Yamaha at first and completed the afternoon in seventh, with his team-mate James Toseland ninth between the two Suzukis.
The only accident of the session was Yuki Takahashi's violent high-side at the halfway point. The Scot Honda rider, who already has a fractured finger from his first lap crash at Catalunya, limped away from the scene with assistance from the marshals.
Pos Rider Bike Time Gap
1. Randy de Puniet LCR Honda 1m37.842s
2. Valentino Rossi Yamaha 1m37.847s + 0.005s
3. Casey Stoner Ducati 1m37.877s + 0.035s
4. Jorge Lorenzo Yamaha 1m38.061s + 0.219s
5. Andrea Dovizioso Honda 1m38.092s + 0.250s
6. Dani Pedrosa Honda 1m38.194s + 0.352s
7. Colin Edwards Tech 3 Yamaha 1m38.205s + 0.363s
8. Chris Vermeulen Suzuki 1m38.350s + 0.508s
9. James Toseland Tech 3 Yamaha 1m38.379s + 0.537s
10. Loris Capirossi Suzuki 1m38.429s + 0.587s
11. Alex de Angelis Gresini Honda 1m38.495s + 0.653s
12. Nicky Hayden Ducati 1m38.928s + 1.086s
13. Mika Kallio Pramac Ducati 1m39.464s + 1.622s
14. Marco Melandri Hayate Kawasaki 1m39.597s + 1.755s
15. Toni Elias Gresini Honda 1m39.642s + 1.800s
16. Sete Gibernau Hernando Ducati 1m40.151s + 2.309s
17. Yuki Takahashi Scot Honda 1m40.185s + 2.343s
18. Niccolo Canepa Pramac Ducati 1m41.020s + 3.178s
19. Gabor Talmacsi Scot Honda 1m41.380s + 3.538s