Graphene oxide nanomaterials are being developed for wide-ranging applications, but have potential safety concerns for human health. We conducted a double-blind randomised controlled study to determine how inhalation of graphene oxide nanosheets affects acute pulmonary and cardiovascular function. Small and ultrasmall graphene oxide nanosheets at 200 μg/m3 48 or filtered air were inhaled for 2 hours by 14 young healthy volunteers on repeated visits. Overall, graphene oxide nanosheet exposure was well-tolerated with no adverse effects. Heart rate, blood pressure, lung function and inflammatory markers were unaffected irrespective of graphene oxide particle size. Highly enriched blood proteomics analysis revealed very few differential plasma proteins and thrombus formation was mildly increased in an ex vivo model of arterial injury. Overall, acute inhalation of such highly purified and thin graphene oxide nanosheets of nanometre dimensions was not associated with overt detrimental effects in healthy humans. These findings demonstrate the feasibility of carefully controlled human exposures for risk assessment of graphene oxide, and lay the foundations for investigating the effects of other 2D nanomaterials in humans.