ObjectiveMultiple sclerosis (MS) is a degenerative disease of the central nervous system (CNS) characterized by inflammation, demyelination, and axonal damage. It has been hypothesized that hypoxia plays a role in the pathogenesis of MS. This study was undertaken to investigate the reproducibility of non‐invasively measured cortical microvascular hemoglobin oxygenation (S_tO₂) using frequency domain near‐infrared spectroscopy (fdNIRS), investigate its temporal pattern of hypoxia in people with MS (pwMS), and its relationship with neurocognitive function and mood.MethodsWe investigated the reproducibility of fdNIRS measurements. We measured cortical hypoxia in pwMS, and the relationships between S_tO₂, neurocognitive function, fatigue, and measures of physical disability. Furthermore, we cataloged the temporal pattern of S_tO₂ measured at 1‐week intervals for 4 weeks, and at 8 weeks and ~1 year.ResultsWe show that fdNIRS parameters were highly reproducible in 7 healthy control participants measured over 6 days (p > 0.05). There was low variability between and within subjects. In line with our previous findings, we show that 33% of pwMS (n = 88) have cortical microvascular hypoxia. Over 8 weeks and at ~1 year, S_tO₂ values for normoxic and hypoxic groups did not change significantly. There was no significant association between cognitive function and S_tO₂. This conclusion should be revisited as only a small proportion of the relapsing‐remitting MS group (21%) was cognitively impaired.InterpretationThe fdNIRS parameters have high reproducibility and repeatability, and we have demonstrated that hypoxia in MS is a chronic condition, lasting at least a year. The results show a weak relationship between cognitive functioning and oxygenation, indicating future study is required. ANN NEUROL 2023;94:1067–1079