CO2 artefact can distort estimate of microbial basal respiration rate in closed chambers

• Basal respiration rates drop on average by 3.4-fold within 24 h. • Ruling out biological causes: O 2 depletion and initial activity peak not responsible for decline. • CO 2 artefact identified: dissolved CO 2 outgases from soil water, skewing results. • Warning: CO 2 artefact can be misinterpreted as high initial respiration. • Recommended standardisation: 24-hour incubation and 10:1 v/w headspace:soil ratio. Soil microbial respiration is a key indicator of microbial activity and carbon cycling. It is commonly measured by CO 2 increase in closed-chamber incubations over time. Although basal respiration rates measured between 1 and 24 h are assumed to be constant, they were observed to vary. Identifying the causes is crucial to ensure accurate measurements and derive recommendations for standard operating protocols. Three experiments were conducted to determine whether temporal variation emerges from biological processes (O 2 depletion or activity flush) or physicochemical causes (CO 2 artefact). Incubation duration (12 time points), headspace-to-soil-ratio (1 g and 5 g soil, corresponding to 20:1 and 4:1 v/w) and pre-incubation times (2, 4, 6, and 10 days) were varied systematically for ten soils (two texture classes, gradient of organic carbon content). Apparent basal respiration per gram soil per hour was on average 3.4-fold higher after 1 h than 24 h. Biological reasons for the observed exponential decline were unlikely. Instead, a soil-exclusion experiment confirmed CO 2 dissolution in water during pre-incubation can cause an initial CO 2 -peak, which can be misinterpreted as high initial respiration. Soil moisture, amount of soil, microbial activity, and chamber closure timing affected the artefact’s magnitude. Standardising incubation duration and headspace-to-soil ratio to 24 h and 10:1 v/w can mitigate the CO 2 artefact bias. This harmonisation is particularly important when comparing basal respiration derived from different laboratories and methods.