Toyota, now the only major manufacturer entered in the top LMP1 category after champions Porsche quit, have only themselves to beat but can take nothing for granted after losing out in 2017 due to unreliability.
The Japanese team are seeking their first win at the 20th attempt to become only the second Japanese marque after Mazda to triumph in the endurance classic.
The number eight TS050 hybrid of Nakajima, Alonso and Switzerland's Sebastien Buemi was followed on the timesheets by the number seven car of Japan's Kamui Kobayashi, Britain's Mike Conway and Argentina's Jose Maria Lopez.
Kobayashi took pole last year in a record 3:14.791 and the track has been shortened by three metres since then for safety improvements.
With the possibility of rain on Thursday, when two more qualifying sessions are scheduled, there is a chance that Wednesday's time will secure pole.
While Nakajima set the pace, Le Mans rookie Alonso completed a mandatory five laps in night conditions in a car that took the trio to victory in the opening race of the season at Spa in Belgium.
The Spaniard is combining a full Formula One and world endurance season and arrived at the French circuit straight from competing in last Sunday's' Canadian Grand Prix for McLaren.
Alonso, who has also won the Monaco Grand Prix as well as Formula One titles, is aiming to complete the 'Triple Crown of Motorport' and needs to win Le Mans and the Indianapolis 500 to do so.
He competed at Indianapolis last year, leading before an engine failure.
Frenchman Stephane Sarrazin was third fastest in the number 17 SMP Racing BR1 non-hybrid car with a lap of 3:19.483 while Brazilian Bruno Senna was fourth in the number one Rebellion R13 entry.
The non-hybrid LMP1 cars are lighter but thirstier, requiring more and longer pitstops than the Toyotas.
Jenson Button, the 2009 Formula One champion, also completed his night laps in the number 11 SMP Racing BR1 that was seventh fastest.
The 86th edition of the race starts at 1300GMT (1500 local) on Saturday with Spain's French Open tennis champion Rafael Nadal the official starter of the 60-strong field.