Background <p>Intraoperative ICGFA use is correlated with lower rates of anastomotic leak in colorectal surgery. This study evaluated the cognitive workload (CWL) associated with the interpretation of indocyanine green fluorescence angiography in colorectal surgery, relative to other operative tasks, in both simulated and actual surgeries.</p> Methods <p>Specialty surgical trainees, fellows and consultants were recruited with consent. Heart rate variability (HRV) and functional near-infrared spectroscopy (fNIRS) were used as physiological indicators of CWL during both a simulated skills lab [sequentially measuring CWL at baseline and during ‘simple’ and ‘advanced’ laparoscopic skills and during operative video interpretation of surgical anatomy and ICGFA] and in theatre during resectional colorectal surgery (monitoring relative CWL during serial operative steps immediately before and after ICGFA).</p> Results <p>22 participants completed simulation testing, with 11 also providing in-theatre data. The majority had minimal/no ICGFA experience. In simulation testing, linear mixed-effects (LME) modelling of the fNIRS data indicated that ICGFA interpretation was not significantly associated with higher CWL versus baseline overall, but this varied by individual with consultants and non-GI surgeons having significantly lower CWL for ICGFA interpretation (<i>p</i> &lt; 0.05). HRV modelling revealed somewhat contrasting parameter findings with elevated CWL during laparoscopic skills being the more robust conclusion. In-theatre testing (10 colorectal surgeons) revealed ICGFA interpretation imposed broadly similar CWL to other inline operative steps, being peak CWL in four individuals by HRC or at least one fNIRS measure with one measure indicating significantly higher CWL than exercised in simulation.</p> Conclusions <p>ICGFA interpretation exhibits broadly similar CWL as other operative steps, in both simulated and in-theatre settings with important differences evident by test context.</p>