Some cars struggle going as slow as 30 mph in 5th and might give better mpg in 4th because the engine is running at a more efficient speed.. Idling when stopped in heavy traffic and at traffic lights uses fuel , which is why stop start systems were developed to save fuel. (When they actually work) . I suspect there wont be much difference between the two in fuel, but the route that has less stopping may also have an advantage of less wear and tear on clutch ,brakes and tyres , and less time breathing in traffic fumes. a constant 40 mph in 5th is about as fuel efficient as it gets.
You could try brimming the tank, run one route for a week or so, and record exactly how much fuel it takes to brim the tank again. Then do the same procedure using the alternative route for the same number of days and compare how much petrol was needed this time. (Brimming means carefully filling the tank until you can actually see petrol in the filler neck )
Another possibility. I believe google maps, and possibly some sat navs, have an option to favour the most fuel efficient route. You can also enter a start time to reflect rush hour traffic conditions. . See what it suggests for your commute. It might even suggest one route as best going to work and a different one when returning home. Or an alternative route you had never thought of.
Edit. Even though you are familiar with the routes it might help to always have a live traffic data apps such as waze running on your phone, .If it detects slower than usual traffic ahead, perhaps a delay, it might divert you onto a better route
