Fresh from his success in last weekend's grand prix, Hamilton got straight down to work and soon achieved the best overall time of the two-day test with a 1m34.136s.
His early morning benchmark remained unbeaten throughout the day and the 2008 F1 world champion also accumulated the most laps of the session, being the only driver to reach triple figures with 118 under his belt.
Toro Rosso's Jean-Eric Vergne emerged as the best of the rest after a late run in the afternoon brought him within 1.421 seconds of Hamilton's pacesetting time.
Kevin Magnussen set the third quickest time for McLaren but had the ignominy of bringing the morning session to an early end as he came to a halt in the gravel at Turn 13 with just under five minutes remaining.
Sergio Perez looked set to continue Force India's trend of being the best of the rest in Bahrain as he made progress in the afternoon and emerged as Hamilton's nearest rival, but the Mexican ultimately finished fourth once Vergne and Magnussen picked up the pace.
Rounding off the top five was Daniel Ricciardo, who failed to set a time before lunch due to an electrical issue, but he soon made up the mileage afterwards and ended up 3.174s behind Hamilton after 65 laps of running.
Bahrain F1 test gallery
Marussia's Jules Bianchi was just 0.006s shy of the Red Bull in sixth and had a productive day completing a total of 93 laps.
Giedo van der Garde made his return to an F1 cockpit and beat Sauber stablemate Sergey Sirotkin with his time of 1m37.623s - 1.4s faster than the time set by his fellow non-race driver on Tuesday. The Dutchman lost two hours of running to early engine problems.
Ferrari had a torrid session and completed just 12 laps.
Fernando Alonso was testing with Kimi Raikkonen's chassis from the Bahrain GP weekend, but the team discovered damage to the car that forced it to stop running early.
Although Raikkonen completed the grand prix in 10th place, his car did take a violent trip over a kerb, which was subsequently modified, during Friday practice.
Marcus Ericsson also hit trouble, albeit much later on as Caterham was forced to bring a premature end to its day when he suffered an ERS electrical issue that could not be fixed in time.
Williams reserve driver Felipe Nasr was in action for the Grove-based outfit and clocked in 64 laps as it tested tyres for Pirelli alongside Mercedes.
Nasr finished ahead of Romain Grosjean at the bottom of the order after the Frenchman endured a difficult day with Lotus.
In a repeat of Pastor Maldonado's day one engine problems, Grosjean could only manage 16 laps before suffering the same fate as his team-mate.
His best time was 9.596s off the pace.
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