Marc Marquez slid off his Repsol Honda after losing the front on corner entry, just before the halfway stage of the 30-minute session.
The rookie title leader then returned to the pits on a scooter for his spare bike. Despite the spill, Marquez remained fastest, having set the quickest time of the weekend just before his fall.
However Marquez is likely to have only one bike for qualifying.
Team-mate Dani Pedrosa did not look happy with the handling of his RCV.
Qualifying 1, for those outside the top ten after third free practice - including double 2013 pole sitter Cal Crutchlow - will now get underway.
Yamaha riders Jorge Lorenzo and Valentino Rossi are using the factory's new seamless shift gearbox for the first time in a grand prix weekend.
Ducati test rider Michele Pirro is again replacing the injured Ben Spies at Pramac. Pirro is back on the development 'lab' bike this weekend. Cardion AB's Karel Abraham is returning from injury.
for pictures from the grand prix...
1. Marc Marquez
ESP Repsol Honda Team (RC213V) 1m 33.912s
2. Jorge Lorenzo
ESP Yamaha Factory Racing (YZR-M1) 1m 34.125s
3. Alvaro Bautista
ESP Go&Fun Honda Gresini (RC213V) 1m 34.418s
4. Valentino Rossi
ITA Yamaha Factory Racing (YZR-M1) 1m 34.447s
5. Dani Pedrosa
ESP Repsol Honda Team (RC213V) 1m 34.826s
6. Andrea Dovizioso
ITA Ducati Team (GP13) 1m 35.081s
7. Cal Crutchlow
GBR Monster Yamaha Tech 3 (YZR-M1) 1m 35.142s
8. Stefan Bradl
GER LCR Honda MotoGP (RC213V) 1m 35.171s
9. Michele Pirro
ITA Ignite Pramac Racing (GP13) 1m 35.215s
10. Andrea Iannone
ITA Energy T.I. Pramac Racing (GP13) 1m 35.250s
11. Aleix Espargaro
ESP Power Electronics Aspar (ART CRT) 1m 35.318s
12. Nicky Hayden
USA Ducati Team (GP13) 1m 35.505s
13. Bradley Smith
GBR Monster Yamaha Tech 3 (YZR-M1) 1m 35.656s
14. Colin Edwards
USA NGM Forward Racing (FTR-Kawasaki CRT)* 1m 35.794s
15. Danilo Petrucci
ITA Came IodaRacing Project (Suter-BMW CRT)* 1m 35.812s
16. Randy De Puniet
FRA Power Electronics Aspar (ART CRT) 1m 35.988s
17. Claudio Corti
ITA NGM Forward Racing (FTR-Kawasaki CRT)* 1m 36.257s
18. Hiroshi Aoyama
JPN Avintia Blusens (FTR-Kawasaki CRT)* 1m 36.477s
19. Hector Barbera
ESP Avintia Blusens (FTR-Kawasaki CRT)* 1m 36.626s
20. Yonny Hernandez
COL Paul Bird Motorsport (ART CRT) 1m 37.218s
21. Karel Abraham
CZE Cardion AB Motoracing (ART CRT) 1m 37.277s
22. Michael Laverty
GBR Paul Bird Motorsport (PBM-ART CRT)* 1m 37.553s
23. Bryan Staring
AUS Go&Fun Honda Gresini (FTR-Honda CRT) 1m 37.718s
24. Lukas Pesek
CZE Came IodaRacing Project (Suter-BMW CRT)* 1m 38.451s
* Standard ECU.
Fastest practice time:
ESP Honda1m 33.975s
Official Misano MotoGP records:
AUS Honda1m 33.138s
Fastest race lap:
ESP Yamaha1m 33.906s
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