Wildcard Popyrin, who had only played one grand slam match before this tournament looked fearless.
Thiem, a French Open finalist previous year, called for the trainer midway through the second set citing general fatigue which looked a result of his five-set opening round win.
Using his big forehand weapon, Popyrin hit nearly double the winners of Thiem, who looked sluggish from the outset.
Despite standing 195cm Popyrin showed impressive speed around the court as he pressed the tiring Thiem.
Popyrin took advantage of his rival's frame of mind, attacking the net to break Thiem and take the opening set 7-5.
Thiem had played out a marathon in the first round of the Grand Slam tournament against Frenchman Benoit Paire that went on past 2 in the night before winning 6-4, 6-3, 5-7, 1-6, 6-3.
'When I won the second set was when that thought really kicked in and that I could actually win the match.
"This is the best thing I have ever experienced in my life", the world No. 155 said.
"It is unbelievable to have everyone behind me tonight".
"Gilles is a quality player and I got him in five [sets], that gives me the belief that I can do it against these top guys".
Thiem was visibly uncomfortable throughout the match and stunned the crowd early in the third set when he retired at 2-0 down, gifting Popyrin a passage into the third round. "They were like, 'should we bleach your hair?' I was like, Sure, why not?"
Bolt broke Simon's serve early in the fifth set, the 29th seed unable to counter Bolt's line-catching groundstrokes, eventually taking out the decider 6-4 with a backhand victor down the line.
"Three years ago I wouldn't have thought I'd still be playing tennis, let alone qualifying and winning main-draw matches", Bolt said this week.
In 2016, battling injury and form, Bolt decided a hiatus from tennis would serve him well, joining his brother's fencing business and playing local football at Murray Bridge in South Australia.
Move over Alex de Minaur, Australian tennis has a new teenage tennis sensation in town.
Bolt is coached by former Australian pro Jaymon Crabb, who said the "writing had been on the wall" for his apprentice, noting his strong performances in 2018.