Recording of action potentials from single unmyelinated nerve fibers by microneurography is an important tool to investigate peripheral neural functions in human neuropathies. However, the interpretation of microneurography recordings can be difficult because axonal membrane potential is not revealed by this method. We tested the hypothesis that the recovery cycle of excitability after a single action potential is correlated with changes in the axonal membrane potential. To this end, we used the threshold tracking technique to study how different chemical mediators, with known effects on the membrane potential, influence the post-spike superexcitability of C-fiber compound action potentials in isolated rat sural and vagus nerves. We found that: (1) some chemical mediators (e.g., adenosine 5'-triphosphate) produce a reduction or loss of superexcitability together with increased axonal excitability, indicating membrane depolarization; (2) blockade of axonal hyperpolarization-activated (Ih) currents produces an enhancement of superexcitability together with a decreased excitability, indicating membrane hyperpolarization; and (3) application of calcium produces an increase in membrane threshold without an alteration in superexcitability, indicating a non-specific increase in surface charge and a change in the voltage-dependent activation of sodium channels. In addition, we demonstrated that membrane depolarization and hyperpolarization induce opposite post-spike latency shifts (changes in supernormality) in rat and human nerve segments. Thus, recordings of post-spike excitability and shifts in latency are sensitive techniques for detection of various types of neuromodulation, which are correlated with changes in membrane potential of unmyelinated peripheral axons and may help to understand observations obtained by microneurography in peripheral human neuropathies.