Abstract Complex human physiological processes create the stable isotopic composition of exhaled carbon dioxide (eCO₂), measurable with noninvasive breath tests. Recently, isotope-selective breath tests utilizing natural fluctuation in ¹⁸O/¹⁶O isotope ratio in eCO₂ have been proposed for screening prediabetic (PD) individuals. It has been suggested that ¹⁸O/¹⁶O fractionation patterns reflect shifts in the activity of carbonic anhydrase (CA), an enzyme involved in the metabolic changes in the PD state. To evaluate the applicability of the breath sampling method in Finnish PD individuals, breath delta values (BDVs, ‰) of ¹⁸O/¹⁶O (δ ¹⁸O) were monitored for 120 min in real-time with a high-precision optical isotope ratio spectrometer, both in the fasting state and during a 2 h oral glucose tolerance test (2 h OGTT) with non-labeled glucose. In addition, the BDV of ¹³C/¹²C (δ ¹³C) was measured, and total erythrocyte CA activity was determined. δ ¹⁸O and CA did not demonstrate any statistically significant differences between PD and non-diabetic control (NDC) participants. Instead, δ ¹³C was significantly lower in PD patients in comparison to NDCs in the fasting state and at time points 90 and 120 min of the 2 h OGTT, thus indicating slightly better potential in identifying Finnish PD individuals. However, overlapping values were measured in PD participants and NDCs, and therefore, δ ¹³C cannot be applied as a sole measure in screening prediabetes at an individual level. Thus, because the combination of environmental and lifestyle factors and anthropometric parameters has a greater effect on glucose metabolism and CA activity in comparison to the PD state, ¹⁸O/¹⁶O and ¹³C/¹²C fractionations or CA activity did not prove to be reliable biomarkers for impaired glucose tolerance in Finnish subjects. This study was conducted under the clinicaltrials.gov ID NCT03156478.