The 39-year-old struggled badly in the heat and humidity in Ohio during his match against Argentina's world No. 50 Thiago Agustin Tirante. Djokovic eventually fell 2-6, 6-4, 6-4, despite taking the opening set comfortably.
The defeat marked a disappointing return to competitive action for Djokovic, who was playing his first match since his Wimbledon semi-final loss to Jannik Sinner in July. The Serbian star, currently ranked world No. 5, appeared visibly uncomfortable as the match progressed.
Djokovic's physical struggles became particularly apparent during the second set. He dropped to his hands and knees before taking a medical timeout, raising concerns about whether he would be able to complete the contest.
Although he managed to continue, his level dropped significantly as Tirante took control of the match and eventually completed one of the biggest victories of his career.
Speaking after the defeat, Djokovic congratulated his opponent but admitted that the match had been extremely difficult from a personal standpoint.
"Congrats to my opponent. It was not an enjoyable match for me, for sure. The way I felt… Not the first and I guess not the last time, but it is what it is."
The 24-time Grand Slam champion then provided more insight into the issue affecting him, explaining that it is not a completely new problem.
"It's just a condition that I have, health-wise, that has been bothering me for the past couple of years."
Djokovic suggested that hot and humid conditions can make the symptoms considerably worse, something that appeared to be the case during his encounter with Tirante.
"[It causes] a lot of issues, especially when it is humid and it is hot."
The veteran admitted that he had anticipated the difficult weather conditions before stepping onto the court, but the physical effects were compounded by the mental pressure associated with returning to competition.
"I did anticipate it, but there are all these things, like nerves and everything involved that make it worse, and that's what happened."
Despite the setback, Djokovic did not indicate that he was ready to walk away from Cincinnati or from competitive tennis altogether. However, when asked whether he expected to return to the tournament next year, his response was notably uncertain.
"I certainly hope so, but it looks more likely not, unfortunately. But let's see what the future brings."
The defeat represents another unexpected setback for Djokovic during what has been a challenging period of the season. Having reached the Wimbledon semi-finals, expectations were high for the former world No. 1 as he returned to action in Cincinnati.
Instead, Tirante produced a major upset by recovering from a set down and taking advantage of Djokovic's physical difficulties. For Djokovic, the immediate priority will likely be managing the health issue that has affected him for several years and determining how best to approach the remainder of the season.
At 39, Djokovic remains one of the most accomplished players in tennis history, but his latest comments underline the physical challenges that can accompany competing at the highest level late in a remarkable career. The Cincinnati defeat also leaves questions over his next tournament appearance and whether his health will influence his schedule going forward.