Introduction Proteins are among the basic most abundant bio macromolecules, which involve in various biological functions, as building blocks in biological structure, enzymes and hormones (Chen et al. 2015; Nelson et al. 2008). Protein denaturation and unfolding can lead to a number of chaos in the protein science, food and biopharmaceutical industry. Protein aggregation which results from the non-native folded proteins under the influence of various environmental and intrinsic factors like thermal, shear, ionic strength and molecular crowding. The aggregation of proteins leads to the formation of insoluble β-sheet rich fibrils (Pandey et al. 2012; Solá et al. 2006), which are associated with different amyloidogenic diseases like Parkinson’s disease, Huntington’s disease (neurodegenerative diseases) and also diabetes mellitus (systemic diseases) (Chiti and Dobson 2017; Lansbury and Lashuel 2006; Ross and Poirier 2004).Generally, a protein in a solution is stabilized by different interactions among the amino acid residues, which include the van der Waals, hydrogen bond, hydrophobic interaction, electrostatic interaction and disulphide bridges. These interactions favour the native compact conformation (Anand et al. 2011; Sprague-Piercy et al. 2020). The aggregation of proteins initiates by forming disordered structure in the presence of adverse factors like high temperature, extreme pH, and mechanical stress. The external energy from these factors breaks intramolecular interactions. Thus, native conformation and its original compact conformation slowly deteriorate, leading to a more flexible and open structures, which expose the hydrophobic patches and other free disulphide bonds. This finally renders the intermolecular interactions through these exposed regions leading to the formation of protein aggregates (Bratko et al. 2007; Vetri et al. 2007; Wälchli et al. 2020).Among the various proteins, Bovine serum albumin (BSA) also called as “Fraction V” is the most significant serum protein physiologically, which has a multi diversified function, be it as a carrier molecule and stabilizer. It comprises of three domains with each having two subdomains and is intricately stabilized by 17 intermolecular disulphide bonds excluding one free thiol residue Cys34 (Murayama and Tomida 2004). BSA has a molecular weight around ̵̴ 66 kDa and is majorly constituted of helical secondary structure (Pandey et al. 2013). These features make BSA a model protein for various biophysical and biochemical studies (Chen et al. 2015). Apart from physiological importance, BSA is one of the main components of the whey protein, which is majorly used as gelling agent, emulsifier or foaming agent and therefore the quality of these agents wholly depends on the processing conditions. For instance, the extrusion process in the process biotechnology involves both thermal and mechanical treatments of the protein formulations (Quevedo et al. 2020). Therefore, the knowledge of how this protein behaves under a certain external force or stimuli is essential to decide the processing parameters and finally the desired product (Rondeau et al. 2010).In this direction, effects of various thermal and mechanical stimuli on the properties of proteins have been investigated. Various heat stability studies had been done by researchers in different temperature conditions and found that BSA almost retains its native conformation till 40-50°C range and at around 52-60°C. It shows irreversible unfolding after going through some amount of denaturation. As the temperature increases, approximately above 60°C prominent unfolding and denaturation was obtained resulting in aggregation (Bekard et al. 2012; Murayama and Tomida 2004; Yamasaki et al. 1990). Melting temperature of BSA is reported to be about 63°C (Pal et al. 2020). In another study by Arakawa et al. , BSA was exposed to thermal stressed at 50, 60 and 70°C and the aggregation was checked through native gel electrophoresis and circular dichroism (CD). It was found that no aggregates were formed at 50°C as there were only monomer and dimer bands and in the other cases the monomer band was reduced and few new aggregate bands were observed (Arakawa and Kita 2000).Mechanical impact like shearing or extreme pressure also denatures the conformation of proteins and its formulations (Belitz and Grosch 2013; Quevedo et al. 2020). Proteins have been found to denature under such mechanical force through physically rupturing of the intramolecular interactions. It has been proposed that under a fluid shear a protein undergoes denaturation through two critical steps; firstly, the unfolding protein molecules and secondly the collision between them leading to the aggregation. This ultimately forms larger particles (Anema and McKenna 1996). Various studies have explored the effect of shear on the aggregation of proteins. Stirring, shaking, mechanical agitation and ultrasonication were found to affect the fibril formation (Collins et al. 2004; Grigolato and Arosio 2019; Liu and Lindquist 1999; Maruyama et al. 2001; Serio et al. 2000). However, in most of the practical applications like fluid flow, bioprocessing, formulations, mixing and transportations, thermal and mechanical stresses are simultaneously experienced (Bogahawaththa and Vasiljevic 2020). Thus, it is important to explore thermomechanical behaviour of a protein to grain better insights of the aggregation process.In this regard, thermodynamic aspects of the thermomechanical treatment of BSA and its related impact on the aggregation behaviour have been explored in the present study. The insights associated with the dissipation energy generated during the shearing process and its implications towards the unfolding and aggregation have been investigated. To understand the thermal stability, a hysteresis temperature loop scans of BSA in solution at physiological pH (7.4) were conducted in the three temperature ranges i.e. 25-50-25°C, 25-65-25°C and 25-75-25°C at the ramping rate of 1°C/min. Next, shear induced aggregation of the BSA solution at a constant shear rate of 300 s-1 was conducted at the three temperature conditions (55, 60 and 65 °C) using a MCR 72 Rheometer. To investigate the aggregation behaviour Thioflavin T (Th-T) fluorescence assay was performed to monitor the aggregation kinetics, hydrodynamic diameter was measured using dynamic light scattering (DLS). Far-UV CD was conducted in the range of 190-260 nm to analyze the structural conformational changes. Morphological analysis was performed using atomic force microscopy (AFM).