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I have a custom PCB with an Atmega 32u4 chip. I program the chip with AVRDUDE via ISP after it is has been mounted onto the PCB. All is well.

I now want to add wireless capability via the NRF24L01. The NRF communicates with the 32u4 via ISP. Can I still program the 32u4 on the PCB after the NRF has been mounted or will there be a problem since they are both connected to the ISP pins?

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Yes, that is possible and I've done the exact same thing with an ATmega328 which controls a DAC via SPI. If it wasn't possible ISP wouldn't be an option whenever SPI is already in use. You should make sure that the NRF's CS pin is high while flashing the firmware, otherwise it might corrupt a checksum verification or memory readout.

Note the difference between ISP (in-system programming) and SPI (serial peripheral interface). Even though ISP uses SPI and they have similar abreviations they are not the same.

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