Aims Gas exchange measurements on individual plants depend largely on chamber systems, and uncertainties and corrections in current flux calculation procedures require further assessment. Methods We present a practical study with novel methods for analyses of flux uncertainties in an original chamber design excluding soil fluxes and allowing simultaneous measurements of whole-plant photosynthesis and transpiration. Results Results indicate that random errors caused by IRGA noise and the lack of criteria to optimize the time window (TW) of chamber enclosure lead to significant flux uncertainties (12 %). Although enclosure should be rapid to minimize plant disturbances, longer TWs (3 min) increase confidence in flux estimates. Indeterminate stabilization periods in existing calculation protocols cause significant systematic errors. Stabilization times were identified via the change-point detection method, and flux uncertainties were reduced. Photosynthesis was overestimated by up to 28 % when not correcting the evolving CO2 molar fraction for water vapour dilution. Leakage can compromise flux estimates, but was negligible (ca. 2 %) here due to the large chamber-headspace and relatively small values of both collar contact length and closure time. Conclusions A bootstrapping, resampling-based flux calculation method is presented and recommended to better assess random errors and improve flux precision. We present practical recommendations for the use of whole-plant chambers.