Neostigmine test (NT) is a pharmacological test, demonstrating a clinical improvement in patients affected by myasthenia gravis (MG). We aim to compare clinical evaluation and neurophysiological recordings by concentric-needle single-fiber electromyography (CN-SFEMG) in response to acute administration of neostigmine in ocular and generalized MG patients. Twenty-three MG patients (10 with ocular MG and 13 with generalized MG) were evaluated before and after 90 min neostigmine 0.5-mg administration. Clinical responsiveness was assessed by MG composite (MGC) scale. Neurophysiological evaluation by CN-SFEMG considered analysis of mean value of consecutive differences (MCD), single-pair jitter, and blocks. MGC scores significantly improved after NT in generalized MG patients (MGC 11.1 ± 7.6 vs 9.1 ± 6.7, p = 0.02), whereas the improvement was not significant in the ocular group. CN-SFEMG recordings significantly improved after NT in generalized MG patients (MCD 58.9 ± 18.8 vs 45.9 ± 23.2 μs, p = 0.003; single-pair jitter 49.8 ± 26.9 vs 24.1 ± 26.7%, p = 0.0001; blocks 6.2 ± 9.5 vs 2.6 ± 7.4%, p = 0.03) as well as in ocular MG patients (MCD 50.8 ± 22.7 vs 40.1 ± 22.9 μs, p = 0.01; single-pair jitter 35.9 ± 23.7 vs 20.0 ± 25.1%, p = 0.001). CN-SFEMG is a reliable tool to evaluate responsiveness to acute administration of neostigmine in MG. Moreover, neurophysiological modifications to NT could show subclinical improvement in ocular MG better than that of the clinical scale.