BackgroundOne of the common causes of suboptimal control of thyroid stimulating hormone (TSH) in levothyroxine-treated hypothyroidism is coadministration of proton pump inhibitors (PPIs). Morning administration of pantoprazole has been shown to suppress intragastric pH to a greater extent. We therefore aimed to determine the effect of pantoprazole at different time points of the day on thyroid function test (TFT) in levothyroxine-treated overt primary hypothyroidism.MethodsIn this single centre, hospital based, prospective, two arm cross-over study (AB, BA), participants were randomized into 2 groups based on morning (6:00 am – 7:00 am simultaneously with the scheduled levothyroxine tablet) (group M) and evening (30 min before dinner) intake of 40 mg pantoprazole tablet (group N). After the initial 6 weeks (period 1), a washout period of 1 week for pantoprazole was given, and then both the groups crossed over for another 6 weeks (period 2). Patients were instructed to continue the same brand of levothyroxine tablet at empty stomach 1-hour before breakfast. Serum TSH was measured at baseline, week 6, and week 13.ResultsData from 30 patients, who completed the study with 100% compliance, were analysed. Mean TSH values of the study participants were significantly higher both at week 6 and week 13 compared to the baseline. Mean baseline serum TSH concentrations for groups M and N were 2.70 (± 1.36), and 2.20 (± 1.06) µlU/mL, respectively. Mean serum TSH concentrations at the end periods 1 and 2 for group M were 3.78 (± 4.29), and 3.76 (± 2.77) while the levels in group N were 3.30 (± 1.90), and 4.53 (± 4.590) µlU/mL, respectively. There was a significant rise in serum TSH concentration across periods 1 and 2 in both the groups (F2, 58= 3.87, p = 0.03). Within group changes in TSH across periods 1 and 2 were not statistically significant. Similarly difference in TSH between the groups, either at 6 weeks or at 13 weeks, were also not statistically significant.ConclusionsConcomitant use of pantoprazole, even for 6 weeks, leads to significant elevation in serum TSH in levothyroxine-treated patients who are biochemically euthyroid, irrespective of timing of pantoprazole intake. Early morning and night-time administration of pantoprazole have similar effect on TFT in these patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13044-023-00156-6.