α-Synuclein is an intrinsically disordered protein whose aggregation is associated with Parkinson's disease and other related neurodegenerative disorders. Recently, two single-domain camelid antibodies (nanobodies) were shown to bind α-synuclein with high affinity. Herein, we investigated how these two nanobodies (NbSyn2 and NbSyn87), which are directed to two distinct epitopes within the C-terminal domain of α-synuclein, affect the conformational properties of this protein. Our results suggest that nanobody NbSyn2, which binds to the five C-terminal residues of α-synuclein (residues 136-140), does not disrupt the transient long-range interactions that generate a degree of compaction within the native structural ensemble of α-synuclein. In contrast, the data that we report indicate that NbSyn87, which targets a central region within the C-terminal domain (residues 118-128), has more substantial effects on the fluctuating secondary and tertiary structure of the protein. These results are consistent with the different effects that the two nanobodies have on the aggregation behavior of α-synuclein in vitro. Our findings thus provide new insights into the type of effects that nanobodies can have on the conformational ensemble of α-synuclein.