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The dataset was collected using the leaf chamber spectrometer connected to an infrared greenhouse gas analyser (Li-6400) on soybean (<i>Glycine max</i> L. Merr.) cultivar (Williams 82) third trifoliate leaves on 3-week old plants (vegetative growth stages V3 and V4). Seeds were sourced from Iowa State University, USA. Plants were grown in pots in the controlled environment facility in the Faculty of Biological Sciences, Plant Growth Suite, University of Leeds. The growth conditions were 25C day/night temperatures, 50% relative humidity, with a 16 h photoperiod at an irradiance of 400–600 μmol photons /m²/s.