Henderson posted a time of 7 minutes 23 seconds around the 6.4km circuit on the roads of the Belgian city of Liege, 20 seconds behind stage winner and time trial expert Fabian Cancellara of Switzerland.
Henderson, competing in his first Tour de France, has been included in the race by his Lotto-Belisol team for his sprinting ability.
He will be expected to earn his keep on the fast and flat early stages when leading out sprinter Andre Greipel, who finished 20 places behind Henderson in the prologue.
The 35-year-old Henderson, the best prologue place-getter in his team, tweeted afterwards: "That was a long 7.5 minutes."