Don't worry: Of course it saves the count and thus len() on lists is a pretty cheap operation. Same is true for strings, dictionaries and sets, by the way!
Write your program so that it's optimised for clarity and easily maintainable. Is your program clearer with a call to len(foo)? Then do that.
Are you worried about the time taken? Use the timeit module in the standard library to measure the time taken, and see whether it is significant in your code.
You will, like most people, very likely be wrong in your guesses about which parts of your program are slowest. Avoid the temptation to guess, and instead measure it to find out.
Remember that premature optimisation is the root of all evil, in the words of Donald Knuth. Only focus on the speed of code that you have measured the speed of, to know whether the benefit would be worth the cost of changing how it works.
I'm quite late to the party, but I just did a test and got some interesting results:
import timeit, statistics
test_arr = list(range(1000000))
# Just call len() every time
def test1(arr):
for i in range(len(arr)):
j = arr[len(arr)-1]
# Store the result of len()
def test2(arr):
l = len(arr)
for i in range(len(arr)):
j = arr[l-1]
print("Running test 1...")
t1 = timeit.repeat(lambda: test1(test_arr), number=int(2e2), repeat=4)
print("Test 1 results:")
print(t1)
print("Running test 2...")
t2 = timeit.repeat(lambda: test2(test_arr), number=int(2e2), repeat=4)
print("Test 2 results:")
print(t2)
m1 = statistics.mean(t1)
m2 = statistics.mean(t2)
avg = round(100 * abs(m2-m1)/m1, 2)
sign = "quicker" if m1-m2 > 0 else "slower"
print(f"On average, test 2 was {avg} {sign} than test 1")
Results:
Running test 1...
Test 1 results:
[15.189714099979028, 15.160469999769703, 15.17295220005326, 15.072236399864778]
Running test 2...
Test 2 results:
[8.17264029989019, 8.191439799964428, 8.171442999970168, 8.208112000022084]
On average, test 2 was 45.96 quicker than test 1
It's a very crude test and I'm sure somebody can improve upon it (or point out a mistake), but it appears that it is indeed significantly quicker to call len only once and save it to a variable, rather than calling it repeatedly (which I believe is what the poster was asking).