Metabolism and energetics research have contributed to improving and understanding livestock production efficiency and its associated impacts on the environment. Open-circuit respiration chambers are used to calculate an animal's heat production and/or emission rate from a combination of respiratory gas exchange, ventilation rate, temperature, and relative humidity measurements. The whole system must be evaluated before application to assess function, integrity, and identify sources of measurement error through a mass recovery (MR) test, which compares the mass measured by the system to a known mass (reference) injected inside the chamber. The objectives of this work were to compare the calculated MR percent and associated standard uncertainty for four reference injection approaches: (1) mass flow control (MFC) of SF6 (tracer gas; TG) and (2) CH4, (3) gravimetric analysis (GA) of SF6, and (4) MFC of SF6 with a diffuser. Three MR tests were conducted in three chambers each, for every approach. Impact of injection location and time to reach the fresh air exchange steady-state was assessed by addition of the diffuser (approach 4) and was found to be ∼2 min faster than using a single outlet used in MFC of SF6 and CH4. Significant differences for Chamber 1 (P- 0.002) and Chamber 2 (P = 0.02), but not Chamber 3 were found using a one-way ANOVA comparison, which relies on the variance of the measured MR percent, and does not incorporate measurement error. Further, when standard uncertainty was included, no significant differences between injection approaches were found. This was attributed to the large standard uncertainty associated with the injection approaches, ranging from 3.0% to 14.7%. A well-documented example of performing a MR test and associated uncertainty analysis is needed for understanding the impact of equipment selection (e.g. TG concentration, scale resolution and accuracy) and the number of MR test conducted on whole system verification and uncertainty.