Andrea Iannone was once again the quickest rider in practice for the Dutch TT at Assen, though the competition began to close in on the Ducati's – which had dominated opening practice, filling four of the top five positions.
Iannone pipped Yamaha's Valentino Rossi by just 0.004s to go quickest, but was the only Ducati rider in the top five in FP2 as championship leader Marc Marquez found some pace, getting to within two-tenths of the top spot to go third.
Suzuki's Maverick Vinales was fourth, three-tenths back, followed by Yamaha's Jorge Lorenzo to complete the top five, all split by just four-tenths of a second.
Danilo Petrucci continued his impressive form to secure seventh behind Andrea Dovizioso, beating brothers Aleix Espargaro (Suzuki) and Pol Espargaro (Tech 3) as well as his team-mate Scott Redding, tenth.
Avintia Ducati's Hector Barbera crashed out after suffering a lowside at Turn 3, which was the same location Alvaro Bautista lost control of his Aprilia bike after just a few minutes of the session starting.
Bautista suffered a second crash, this time at Turn 5. Aspar Ducati's Eugene Laverty got caught up in the accident.