Final answer:
During the refractory period, inactivated sodium channels and the efflux of potassium ions make it harder to generate a second action potential.
Step-by-step explanation:
The refractory period following an action potential makes it harder to generate a second action potential soon after the first one. This is because during the refractory period, some voltage-gated Na+ channels are inactivated, preventing the generation of a new action potential. Additionally, the efflux of K+ ions opposes depolarization toward the threshold, making it more difficult to reach the necessary membrane potential for another action potential. Therefore, option 4) - all of these factors - make it harder to generate a second action potential.