See the Programming Dialog pages in the Atmel Studio help file for information on the STK600 programming dialog.
The pinout of the 6- and 10-pin ISP headers are shown below:
It is not necessary to remove the ISP cable while running a program in the AVR device. The port pins used for ISP programming can be used for other purposes in the user's program.
See also section In-System Programming of an External Target System.