The side lost 25-24 to the Rebels in Melbourne last night, conceding a try to Rebels winger Dom Shipperly right on fulltime.
The Highlanders had looked safe for a victory when up 24-13 up with a few minutes to go, but big Rebels centre Mitch Inman scored a try and then Shipperly managed to get over to bag the win.
The Highlanders had previously beaten the Crusaders and Brumbies in pre-season games.
The match was a free-flowing affair and both sides made multiple team changes throughout the match which was played in four quarters.
It took place at the Harlequins Rugby Club, a suburban ground in Melbourne, the first time a match between professional teams has been played on a club rugby ground in Melbourne.
The Melbourne side started the better and scored the first try through young fullback Jonah Placid.
Lima Sopoaga had scored the opening points of the game - a penalty - after Highlanders pressure but the Rebels answered with the score from Placid, a Queensland product.
Former New Zealand colt Mike Harris banged over the conversion and added a penalty soon after.
The Highlanders then scored a five-pointer through big winger Patrick Osborne after some good lineout play by the forwards.
Sopoaga could not add the extras from far out and Harris managed a penalty to have his side ahead 13-8 at the break.
Both teams made plenty of changes and one of those was Tasman first five-eighth Marty Banks, who pulled on the Highlanders jersey for the first time, having missed the first two games with injury.
However, he quickly made an impact banging over two penalties which put the visiting side in front.
Highlanders utility back Trent Renata then scored a try well into the final quarter and it looked as though the Highlanders would get home.
The Rebels never gave up and Shipperly's try made it two wins for the franchise in its pre-season after defeating the Reds last week.
The Melbourne side has a tough opener to its Super rugby campaign this year, taking on the Crusaders in Christchurch next Friday night, the opening game of the competition.
The Highlanders will head home today and have the weekend to recover.
The side has a bye in the first week of the competition - not always ideal for a team after a successful pre-season.
The team will train next week and then have a game among themselves towards the end of the week.
