Despite improvement in the management of modifiable cardiovascular risk factors, ischemic stroke remains the leading cause of morbidity and mortality in the adult population. The aim of this study was to analyze the time-dependent dynamic differences in expression of the nitric oxide (NO) metabolic pathway in the platelet and plasma compartment between subjects with and without ischemic stroke. Additionally, the interplay between these parameters and platelet aggregation was investigated. A total of 418 patients in acute phase of non-cardioembolic stroke were investigated. Following the inclusion and exclusion criteria, finally 40 subjects with stroke and 39 demographically matched healthy participants were enrolled. Neurological physical examination, followed by assessment of the platelet and plasma levels of the nitric oxide synthase (NOS) inhibitors, including asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), as well as NOS substrate-L-Arginine were performed dynamically three times within the first 24-h, then on the 3rd and 7th day after the stroke onset, which was compared with the healthy control. The platelet L-Arginine concentration was significantly higher on the 1st and 3rd day of stroke, while the plasma levels were significantly lower on exact days in comparison to the control. The competitive NOS-inhibitors in platelets were stably elevated in stroke subjects, whereas no significant differences in plasma compartment were noted. The arachidonic-acid-induced platelet aggregation was negatively associated with the platelet NOS substrate bioavailability, as assessed by the L−Arginine ADMA-ratio on the 3rd and 7th day. Subjects with non-cardioembolic ischemic stroke are characterized by elevated platelet levels of NOS inhibitors. Management of stroke results in increasing the platelet L-Arginine concentration and subsequent NO bioavailability in the platelet compartment.