Hughes started strongly, posting the third best time at the first time check as she looked to close out her Olympic career with a medal. However at the second check after 20.4 kilometres, the experienced Canadian had slipped two places to fifth, where she would remain at the finish.
"I had everything I needed, the best bike, and all the support from my federation. That was the best I had. There's nothing could have done more, I just wasn't good enough. This was my big goal to prepare for, and I prepared better than I ever had for this and the best I had was fifth place. Do I have sense of disappointment that I got to represent my country at the Olympics in two different events and at my sixth Olympics? Absolutely not It's a gift - an honour," she said at the finish in Hampton Court.
Due to turn 40 next month, Hughes will carry on competing to at least the end of this season for her Specialized - Lululemon trade team, and she has set the Worlds team time trial as another major objective.
Asked if this would be her last Games, she said, "I'm pretty sure, yeah. I mean, ask me tomorrow but I've known that going through these Games that this was probably the last chance I'd have to do this in my life and it really made me happy to be here to have my chance."
It was rival Kristin Armstrong (United States of America) who sealed the gold medal, retaining her title from four years ago in Beijing. Like Hughes, Armstrong has come back from a hiatus from the sport and the Canadian praised Armstrong's tenacity to succeed and overcome challenges.
"Kristin is an awesome athlete. I actually covered her for media in Beijing when she won and when she announced her retirement the first thing my husband said was that she'd be back," Hughes said.
"We're all top level athletes and we get back up and push through the pain and Kristin is no different. She's world class and it's phenomenal what she's done. I had probably had the best time trial I've ever had and I was fifth place. It's phenomenal."