The Sir Michael Stoute-trained mare galloped into history in the hands of Ryan Moore, becoming the first horse since the race was first staged over 200 years ago to win the two-and-a-half-mile feature in the colours of a reigning monarch.
She was unable to make a significant impact on her only subsequent start in 2013, finishing seventh in Ascot's Long Distance Cup on Champions Day in October, and has not been seen in competitive action since.
Connections freely admit Estimate's early-season preparation for the defence of her crown has not been ideal, although confidence appears to have grown in recent weeks and the five-year-old thrilled in her work on Saturday morning.
The Queen's racing manager John Warren said: "Estimate had a slow start in her preparation due to some tweaked muscles that prevented her from running in the Sagaro Stakes.
"It has been a little frustrating not to get a prep race into her, but she's trained by Sir Michael Stoute and if there is anyone who can get her there without the benefit of a prep race he's the man to do it.
"In my mind I've got her finishing third, there are two other very good horses in there, but if the ground keeps drying out - she likes top of the ground - and if it helps her maybe use her stamina better than the others, then things can happen.
"Sir Michael seems upbeat and confident that she can put up a bold show. If she shows the same form as last year, and as she's a year older she might improve a pound or two, then hopefully she can be right up there."
The horse most likely to upset the Royal celebrations is the Aidan O'Brien-trained Leading Light. The four-year-old has won six times from just eight career starts, claiming the Queen's Vase at last year's Royal meeting before going on to clinch Classic glory in the St Leger at Doncaster.
He appeared better than ever when making a winning return in the Vintage Crop Stakes and is favourite to give O'Brien a sixth Gold Cup victory following extraordinary four-time winner Yeats and 2011 hero Fame And Glory.
A win for Mick Winters with Missunited would surely only be second in popularity to a win for Estimate, but the gallant former Galway Hurdle heroine faces a big step up in class.
"She's in great order. It was an ambition of the owners for her to run in this, so at least we've got her there," said the County Cork trainer.
"She's coming in off the back of a good win last time and will have improved a fraction for that, whether she's improved enough to get involved I don't know. She's probably at her peak for the year and if she finishes in the money I'd be delighted, we can't expect more than that.
Andrew Balding's Havana Beat is another at a big price but the trainer feels he may outrun his odds.
"He wants fast ground and to be honest, I know he's a 50-1 shot, but he's a pretty high-class horse on his day," said Balding. "He should have won the Bahrain Trophy last year and he was over the top and the ground was too slow for him in the St Leger. He's a very handy horse and should stay well."