Yes, all file download links sent in purchase receipts expire after a set amount of time. Once a download link is expired, it cannot be used again.
By default, download links are valid for 24 hours. The expiration time can be adjusted from the Downloads → Settings → Misc → File Downloads page:
Simply set it to the number of hours you wish download links to be valid for.
Why do download links expire?
The purpose of this feature is to prevent someone from sharing their download links in a public forum. If someone were to share their download link, anyone could use it, but only until it expires (default is 24 hours). This prevents long-term piracy that can harm your store, while also keeping the process simple for normal customers. Once a download link is expired, the customer needs to get a fresh download link if they wish to download. They can do this by logging into their account, or you can re-send their purchase receipt email to them on the Order screen in wp-admin.
It’s important to note that download links are different than the ability to download. An expired download link will not prevent the customer from being able to download their product entirely. A single download link will expire, but if the customer logs in and attempts to download, a fresh download link will be generated for them again. That fresh download link will expire in 24 hours.
To learn more about the ways you can protect your files in Easy Digital Downloads, see the doc on File Downloads in Easy Digital Downloads.