Rio 2016: Armstrong completes time-trial hat-trick
Armstrong came out of retirement for a second time to defend her title and the veteran American proved she is still the best in the business on Wednesday despite her advancing years.
Having won gold at the 2008 Beijing Games and in London four years ago, Armstrong rose to the occasion yet again in the wind and rain to top the podium.
Armstrong clocked 44 minutes 26.42 seconds over the 29.9 kilometre course, denying Russia's Olga Zabelinskaya by 5.55secs, while Anna van der Breggen of the Netherlands added a bronze medal to the gold she won in the road race on Sunday.
The last rider to go out on a route heavy on climbs, Armstrong made history by becoming the first athlete to win three gold medals in the same road cycling event.
Zabelinskaya, cleared to race in February after accepting an 18 month doping ban, could only watch on as Armstrong put the hammer down in the last section of the route to seal victory.
An emotional Armstrong was exhausted as she dropped to the pavement at Pontal after crossing the line and asked "Did I win?”
Zabelinskaya, winner of road race and time trial bronze at the London Games, had seen Van der Breggen finish almost six seconds slower than her at the end of a challenging, technical course, but Armstrong thrived on the pressure to snatch the title.
Van der Breggen's compatriot Ellen van Dijk was edged out of the medals, while Italy's Elisa Longo Borghini finished fifth.