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Aims: The objectives of this study were to compare techniques for measuring biological nitrogen fixation (BNF) and to assess how fertiliser N input affects the balance between BNF and sustainable herbage production on perennial ryegrass ( Lolium perenne L.)/white clover ( Trifolium repens L.) grassland. Methods: Biological N fixation and herbage production by white clover based grassland was measured in 2011 and 2012 under four nominal annual fertiliser N inputs: 0 (0N), 86 (86N), 140 (140N) and 280 kg ha (280N). Biological N fixation was measured using the N isotope dilution and N natural abundance techniques under all fertiliser N inputs and also using the nitrogen difference technique under 0N. Results: The two N techniques produced similar annual estimates of above-ground BNF across the range of fertiliser N inputs. Fertiliser N input resulted in higher herbage dry matter yield, but reduced annual BNF which averaged 80, 64, 66 and 47 kg ha on 0N, 86N, 140N and 280N, respectively, across both techniques and years. Conclusions: The two N techniques were comparable in estimating BNF. Intermediate fertiliser N inputs achieved a balance between minimising detrimental impact on BNF and potential N loss while producing more herbage DM yield than clover swards receiving no fertiliser N. [ABSTRACT FROM AUTHOR]