As the water requirement for food production and other human needs grows, quantification of environmental flow requirements (EFRs) is necessary to assess the amount of water needed to sustain freshwater ecosystems. EFRs are the result of the quantification of water necessary to sustain the riverine ecosystem, which is calculated from the mean of an environmental flow (EF) method. In this study, five EF methods for calculating EFRs were compared with 11 case studies of locally assessed EFRs. We used three existing methods (Smakhtin, Tennant, and Tessmann) and two newly developed methods (the variable monthly flow method (VMF) and the Q 90 _ Q 50 method). All methods were compared globally and validated at local scales while mimicking the natural flow regime. The VMF and the Tessmann methods use algorithms to classify the flow regime into high, intermediate, and low-flow months and they take into account intra-annual variability by allocating EFRs with a percentage of mean monthly flow (MMF). The Q 90 _ Q 50 method allocates annual flow quantiles ( Q 90 and Q 50 ) depending on the flow season. The results showed that, on average, 37% of annual discharge was required to sustain environmental flow requirement. More water is needed for environmental flows during low-flow periods (46–71% of average low-flows) compared to high-flow periods (17–45% of average high-flows). Environmental flow requirements estimates from the Tennant, Q 90 _ Q 50 , and Smakhtin methods were higher than the locally calculated EFRs for river systems with relatively stable flows and were lower than the locally calculated EFRs for rivers with variable flows. The VMF and Tessmann methods showed the highest correlation with the locally calculated EFRs ( R 2 =0.91). The main difference between the Tessmann and VMF methods is that the Tessmann method allocates all water to EFRs in low-flow periods while the VMF method allocates 60% of the flow in low-flow periods. Thus, other water sectors such as irrigation can withdraw up to 40% of the flow during the low-flow season and freshwater ecosystems can still be kept in reasonable ecological condition. The global applicability of the five methods was tested using the global vegetation and the Lund-Potsdam-Jena managed land (LPJmL) hydrological model. The calculated global annual EFRs for fair ecological conditions represent between 25 and 46% of mean annual flow (MAF). Variable flow regimes, such as the Nile, have lower EFRs (ranging from 12 to 48% of MAF) than stable tropical regimes such as the Amazon (which has EFRs ranging from 30 to 67% of MAF).