The Italian Red Devils Roma Aprilia rider set a time of 1:31.317 under clear skies, putting him 0.075s ahead of Irishman Eugene Laverty on the works Aprilia.
"Conditions were a bit more difficult during the afternoon compared to the morning, due primarily to the wind that made changing direction harder," said Fabrizio.
"Last Monday I was hit by a seagull on my arm while I was on the straight and I am still feeling the pain. I can say though, that we’re heading in the right direction."
Laverty said: “This morning we weren't extremely fast, but in the afternoon we made some changes on my RSV4 to adapt it to the conditions and we took a big step forward.
"My single lap is good, but the key is to maintain consistency for the entire race."
There were five manufacturers represented in the top seven positions, with Marco Melandri (BMW Motorrad GoldBet) third followed by FIXI Crescent Suzuki duo Leon Camier and Chaz Davies, Leon Haslam (Pata Honda) and 2012 championship runner-up Tom Sykes (Kawasaki).
Carlos Checa made it on track for seven laps after his spectacular crash on the Ducati Alstare in first practice.
He was 12th, with the top 13 riders all within a second of pace-setter Fabrizio.
2012 title winner Max Biaggi has retired from racing.
1. Michel Fabrizio (Aprilia) 1'31.317
2. Eugene Laverty (Aprilia) 1'31.392
3. Marco Melandri (BMW) 1'31.539
4. Leon Camier (Suzuki) 1'31.608
5. Chaz Davies (BMW) 1'31.638
6. Leon Haslam (Honda) 1'31.687
7. Tom Sykes (Kawasaki) 1'31.754
8. Davide Giugliano (Aprilia) 1'31.793
9. Sylvain Guintoli (Aprilia) 1'31.863
10. Jonathan Rea (Honda) 1'31.884
11. Jamie Stauffer (Honda) 1'31.984
12. Carlos Checa (Ducati) 1'32.063
13. Max Neukirchner (Ducati) 1'32.243
14. Glenn Allerton (BMW) 1'32.352
15. Loris Baz (Kawasaki) 1'32.497
16. Ayrton Badovini (Ducati) 1'32.540
17. Ivan Clementi (BMW) 1'32.757
18. Jules Cluzel (Suzuki) 1'32.801
19. Federico Sandi (Kawasaki) 1'32.898
20. Vittorio Iannuzzo (BMW) 1'33.082
21. Alexander Lundh (Kawasaki) 1'33.305