<p>This cross-sectional observational study assessed exposure to a selected combustible cigarette (CC) smoke constituent and biomarkers of potential harm (BoPH) in healthy adults who exclusively used a heated tobacco product (direct heating tobacco system, platform 3, generation 3, version a [DT3.0a] group,&#xa0;<i>n</i> = 304), smoked CC (CC group,&#xa0;<i>n</i> = 102), or had never smoked CC (NS group, <i>n</i> = 102), to evaluate biomarker profiles relevant to health risks associated with smoking. Relative to the CC group, exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone was 39.1% lower in the DT3.0a group (<i>p</i> &lt; 0.01), and several BoPH were lower in the DT3.0a group, including 2,3-dinor-thromboxane-B2 (− 19.4%, <i>p</i> = 0.04), 11-dehydrothromboxane-B2 (− 10.2%, <i>p</i> = 0.25), 8-epi-prostaglandin-F2α (− 7.2%, <i>p</i> = 0.42), and white blood cell count (− 11.6%, <i>p</i> = 0.05). Relative to the CC group, high-density lipoprotein cholesterol was 32.0% higher in the DT3.0a group (<i>p</i> &lt; 0.01) with pulmonary function parameters also being higher, although the differences were not statistically significant. For these biomarkers, the NS group exhibited trends similar to those observed in the DT3.0a group. In contrast to previous findings, soluble intercellular adhesion molecule-1 levels were similar between the CC and NS groups and highest in the DT3.0a group, suggesting that further assessments may be more appropriate in the context of change in values. Except for this point, the DT3.0a group showed BoPH profiles trending in the direction toward those of the NS group, suggesting that the health risks associated with smoking are potentially lower in DT3.0a use compared to CC smoking.</p><p><b>Trial Registration :&#xa0;</b>Prior to the recruitment, the study was registered at the UMIN Clinical Trials Registry on December 27, 2022(UMIN000049840).</p>